Process for the Preparation of Compositions for Modulating a Kinase Cascade and Methods of Use Thereof

ABSTRACT

The invention relates to compositions comprising 2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamide and its mesylate and dihydrochloride salts. More specifically, the invention provides an efficient process for the synthesis of 2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamide and its mesylate and dihydrochloride salts and methods for modulating one or more components of a kinase cascade using the compositions of the invention.

RELATED APPLICATIONS

This application claims priority to and is a Continuation application ofU.S. application Ser. No. 12/154,056, filed May 19, 2008, which claimspriority to U.S. provisional patent application Ser. No. 60/930,758,filed May 17, 2007 and is a Continuation In Part of U.S. applicationSer. No. 12/005,792, filed Dec. 28, 2007; and U.S. application Ser. No.12/005,792 claims priority to U.S. provisional patent application Ser.No. 60/877,762, filed Dec. 28, 2006. The entire contents of eachapplication are incorporated by reference herein.

FIELD OF THE INVENTION

The present invention is directed to compositions and processes for thesynthesis of substantially pure2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamide(KX2-391), and its mesylate and bis-hydrochloride salts. The inventionalso relates to methods of using such compositions.

BACKGROUND OF THE INVENTION

Signal transduction is any process by which a cell converts one kind ofsignal or stimulus into another. Processes referred to as signaltransduction often involve a sequence of biochemical reactions insidethe cell, which are carried out by enzymes and linked through secondmessengers. In many transduction processes, an increasing number ofenzymes and other molecules become engaged in the events that proceedfrom the initial stimulus. In such cases the chain of steps is referredto as a “signaling cascade” or a “second messenger pathway” and oftenresults in a small stimulus eliciting a large response. One class ofmolecules involved in signal transduction is the kinase family ofenzymes. The largest group of kinases are protein kinases, which act onand modify the activity of specific proteins. These are used extensivelyto transmit signals and control complex processes in cells.

Protein kinases are a large class of enzymes which catalyze the transferof the γ-phosphate from ATP to the hydroxyl group on the side chain ofSer/Thr or Tyr in proteins and peptides and are intimately involved inthe control of various important cell functions, perhaps most notably:signal transduction, differentiation, and proliferation. There areestimated to be about 2,000 distinct protein kinases in the human body,and although each of these phosphorylate particular protein/peptidesubstrates, they all bind the same second substrate, ATP, in a highlyconserved pocket. Protein phosphatases catalyze the transfer ofphosphate in the opposite direction.

A tyrosine kinase is an enzyme that can transfer a phosphate group fromATP to a tyrosine residue in a protein. Phosphorylation of proteins bykinases is an important mechanism in signal transduction for regulationof enzyme activity. The tyrosine kinases are divided into two groups;those that are cytoplasmic proteins and the transmembranereceptor-linked kinases. In humans, there are 32 cytoplasmic proteintyrosine kinases and 58 receptor-linked protein-tyrosine kinases. Thehormones and growth factors that act on cell surface tyrosinekinase-linked receptors are generally growth-promoting and function tostimulate cell division (e.g., insulin, insulin-like growth factor 1,epidermal growth factor).

Inhibitors of various known protein kinases or protein phosphatases havea variety of therapeutic applications. One promising potentialtherapeutic use for protein kinase or protein phosphatase inhibitors isas anti-cancer agents. About 50% of the known oncogene products areprotein tyrosine kinases (PTKs) and their kinase activity has been shownto lead to cell transformation.

The PTKs can be classified into two categories, the membrane receptorPTKs (e.g. growth factor receptor PTKs) and the non-receptor PTKs (e.g.the Src family of proto-oncogene products). There are at least 9 membersof the Src family of non-receptor PTKs with pp 60^(c-src) (hereafterreferred to simply as “Src”) being the prototype PTK of the familywherein the approximately 300 amino acid catalytic domains are highlyconserved. The hyperactivation of Src has been reported in a number ofhuman cancers, including those of the colon, breast, lung, bladder, andskin, as well as in gastric cancer, hairy cell leukemia, andneuroblastoma. Overstimulated cell proliferation signals fromtransmembrane receptors (e.g. EGFR and p185HER2/Neu) to the cellinterior also appear to pass through Src. Consequently, it has recentlybeen proposed that Src is a universal target for cancer therapy, becausehyperactivation (without mutation) is involved in tumor initiation,progression, and metastasis for many important human tumor types.

Because kinases are involved in the regulation of a wide variety ofnormal cellular signal transduction pathways (e.g., cell growth,differentiation, survival, adhesion, migration, etc.), kinases arethought to play a role in a variety of diseases and disorders. Thus,modulation of kinase signaling cascades may be an important way to treator prevent such diseases and disorders.

A small-scale synthesis of KX2-391 has recently been published(US20060160800A1). This synthesis is impractical for producing largequantities of the compound and the resulting product suffers fromcontamination with ethyl chloride, which is known to be a weakalkylating agent. Thus, the presence of ethyl chloride at sufficientlyhigh levels limits the pharmaceutical effectiveness of KX2-391compositions.

Accordingly, there is a need for an improved synthetic route to KX2-391that is amenable to commercial production, which is safe and simple andwhich produces KX2-391 and its salts on a large scale in high yield andwhich is substantially pure.

SUMMARY OF THE INVENTION

Compounds of the invention are useful in modulation a component of thekinase signaling cascade. Some compounds may be useful in modulation ofmore than one component of a kinase signaling cascade. The compounds ofthe present invention are useful as pharmaceutical agents. The compoundsof the invention may be useful for modulating regulation of a kinasewhich may be involved in a normal cellular signal transduction pathway(e.g., cell growth, differentiation, survival, adhesion, migration,etc.), or a kinase involved in a disease or disorder.

In one aspect the invention relates to a process for preparing2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamidecomprising the steps of:

-   (1) reacting 4-(2-chloroethyl)morpholine with 4-bromophenol to yield    4-(2-(4-bromophenoxy)ethyl)morpholine;-   (2) coupling 4-(2-(4-bromophenoxy)ethyl)morpholine with    6-fluoropyridin-3-yl-3-boronic acid to yield    4-(2-(4-(6-fluoropyridin-3-yl)phenoxy)ethyl)morpholine;-   (3) reacting 4-(2-(4-(6-fluoropyridin-3-yl)phenoxy)ethyl)morpholine    with acetonitrile to yield    2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)acetonitrile;-   (4) converting    2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)acetonitrile to    methyl 2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)acetate; and-   (5) reacting methyl    2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)acetate with    benzylamine to yield    2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamide.

In another aspect, the invention relates to a process for preparing2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamidemesylate comprising the steps of:

-   (1) reacting 4-(2-chloroethyl)morpholine with 4-bromophenol to yield    4-(2-(4-bromophenoxy)ethyl)morpholine;-   (2) coupling 4-(2-(4-bromophenoxy)ethyl)morpholine with    6-fluoropyridin-3-yl-3-boronic acid to yield    4-(2-(4-(6-fluoropyridin-3-yl)phenoxy)ethyl)morpholine;-   (3) reacting 4-(2-(4-(6-fluoropyridin-3-yl)phenoxy)ethyl)morpholine    with acetonitrile to yield    2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)acetonitrile;-   (4) converting    2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)acetonitrile to    methyl 2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)acetate;-   (5) reacting methyl    2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)acetate with    benzylamine to yield    2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamide;    and-   (6) contacting    2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamide    with methane sulfonic acid to yield    2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamide    mesylate.

In another aspect, the invention relates to a process for preparing2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamidemesylate comprising the step of contacting2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamide withmethane sulfonic acid to yield2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamidemesylate.

In another aspect, the invention relates to a process for preparing2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamidecomprising the step of reacting methyl2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)acetate with benzylamineto yield2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamide.

In another aspect, the invention relates to a process for preparing2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamidecomprising the steps of converting2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)acetonitrile to methyl2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)acetate; and reactingmethyl 2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)acetate withbenzylamine to yield2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamide.

In another aspect, the invention relates to a process for preparing2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamidecomprising the steps of reacting4-(2-(4-(6-fluoropyridin-3-yl)phenoxy)ethyl)morpholine with acetonitrileto yield 2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)acetonitrile;converting 2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)acetonitrileto methyl 2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)acetate; andreacting methyl 2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)acetatewith benzylamine to yield2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamide.

In another aspect, the invention relates to a process for preparing2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamidecomprising the steps of coupling 4-(2-(4-bromophenoxy)ethyl)morpholinewith 6-fluoropyridin-3-yl-3-boronic acid to yield4-(2-(4-(6-fluoropyridin-3-yl)phenoxy)ethyl)morpholine; reacting4-(2-(4-(6-fluoropyridin-3-yl)phenoxy)ethyl)morpholine with acetonitrileto yield 2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)acetonitrile;converting 2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)acetonitrileto methyl 2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)acetate; andreacting methyl 2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)acetatewith benzylamine to yield2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamide.

In another aspect, the invention relates to a process for preparing2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamidecomprising the step of reacting 4-(2-chloroethyl)morpholine with4-bromophenol to yield 4-(2-(4-bromophenoxy)ethyl)morpholine.

In another aspect, the invention relates to a process for preparing2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamidecomprising the step of coupling 4-(2-(4-bromophenoxy)ethyl)morpholinewith 6-fluoropyridin-3-yl-3-boronic acid to yield4-(2-(4-(6-fluoropyridin-3-yl)phenoxy)ethyl)morpholine.

In another aspect, the invention relates to a process for preparing2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamidecomprising the step of reacting44244-(6-fluoropyridin-3-yl)phenoxy)ethyl)morpholine with acetonitrileto yield 2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)acetonitrile.

In another aspect, the invention relates to a process for preparing2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamidecomprising the step of converting2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)acetonitrile to methyl2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)acetate.

In another aspect, the invention relates to a process for preparing2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamidemesylate using any of the processes described above for preparingKX2-391 and comprising the step of contacting2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamide withmethane sulfonic acid to yield2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamidemesylate.

In another aspect, the invention relates to a composition comprisingKX2-391 mesylate salt. In another aspect, the invention relates to acomposition, wherein the KX2-391 mesylate salt has a purity greater than98.0% as determined by HPLC. In another aspect, in the composition, theKX2-391mesylate salt has a purity of 99.0%. In another aspect, in thecomposition, the KX2-391mesylate salt has a purity of 99.5%. In anotheraspect, in the composition, the KX2-391mesylate salt has a purity of99.6%. In another aspect, in the composition, the KX2-391mesylate salthas a purity of 99.7%. In another aspect, the composition contains lessthan 2% an impurity selected from ethyl chloride, ethanol, ethylacetate, heptane, anisole, palladium, and combinations thereof. Inanother embodiment, the composition further comprising apharmaceutically acceptable carrier or excipient.

In another embodiment, the invention relates to a method of using thecomposition of the invention for modulating one or more components of aprotein kinase signaling cascade. In one aspect of the method of theinvention, the composition inhibits a kinase selected from a Src familyprotein kinase, focal adhesion kinase, and a tyrosine kinase. In anotheraspect of the method of the invention, the tyrosine kinase is a Srcfamily protein kinase. In another aspect of the method of the invention,the composition is to be administered orally. In another aspect of themethod of the invention, the composition is to be administeredtopically. In another aspect of the method of the invention, thecomponent of the kinase cascade is responsible for the manifestation ofa disease or disorder selected from hyperproliferative disorders,cancers, pre-cancers, osteoporosis, cardiovascular disorders, immunesystem dysfunction, type II diabetes, obesity, hearing loss, andtransplant rejection.

The above description sets forth rather broadly the more importantfeatures of the present invention in order that the detailed descriptionthereof that follows may be understood, and in order that the presentcontributions to the art may be better appreciated. Other objects andfeatures of the present invention will become apparent from thefollowing detailed description considered in conjunction with theexamples.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph indicating the DSC of KX2-391.2HCl lot 02BP111F.

FIG. 2 is a graph indicating the DSC of KX2-391.2HCl lot 02BP111E.

FIG. 3 is a graph indicating the XRPD of KX2-391.2HCl lot 02BP111E.

FIG. 4 is a graph indicating the XRPD of KX2-391.2HCl lot 02BP111F.

FIG. 5 is a ¹H NMR spectrum of KX2-391 (lot 02BP096K).

FIG. 6 is a ¹H NMR spectrum of KX2-391.MSA

DETAILED DESCRIPTION OF THE INVENTION

The details of one or more embodiments of the invention are set forth inthe accompanying description below. Although any methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, the preferred methods andmaterials are now described. Other features, objects, and advantages ofthe invention will be apparent from the description. In thespecification, the singular forms also include the plural unless thecontext clearly dictates otherwise. Unless defined otherwise, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs. In the case of conflict, the present specificationwill control. All publications, patent applications, patents, and otherreferences mentioned herein are incorporated by reference in theirentirety.

Preparation of KX2-391 and its Salts

The synthesis of 4-(2-(4-(6-fluoropyridin-3-yl)phenoxy)ethyl)morpholineis shown in the scheme below:

4-(2-(4-(6-fluoropyridin-3-yl)phenoxy)ethyl)morpholine (5) wassynthesized in 3 steps. Intermediate 2 was synthesized using an ethercoupling reaction e.g., using Williamson ether synthesis. Etherformation between 4-(2-chloroethyl)morpholine (1) and 4-bromophenol wascarried out in the presence of potassium carbonate and DMF to afford4-(2-(4-bromophenoxy)ethyl)morpholine (2). Rigorously dry conditionswere not essential for this reaction and a basic wash with sodiumhydroxide was used to remove any remaining 4-bromophenol. In anotheraspect of the invention, intermediate 2 is synthesized using any etherformation reaction. Intermediate 2 is synthesized starting from compound1 containing any leaving group. For example, the skilled chemist wouldstart with compounds of the general formula:

wherein the leaving group “LG” includes but is not limited to halogen(as indicated in Compound 1), tosylate, mesylate, triflate, etc.

Compound 5 was formed using a Suzuki reaction. Formation of the arylborate, 6-fluoropyridin-3-yl-3-boronic acid (4), was carried out byforming the aryl anion using n-BuLi followed by in situ quenching withtriisopropylborate (Li, et al., J. Org. Chem. 2002, 67, 5394-5397). Theresulting 6-fluoropyridin-3-yl-3-boronic acid (4) was coupled to4-(2-(4-bromophenoxy)ethyl)morpholine (2) in a solution of DME andaqueous sodium carbonate using tetrakis(triphenylphosphine)palladium toafford 4-(2-(4-(6-fluoropyridin-3-yl)phenoxy)ethyl)morpholine (5), whichwas purified using silica gel chromatography. The skilled chemist wouldknow that other transition metal coupling reactions are used to preparecompound 5.

The synthesis of2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamidedihydrochloride is shown below:

2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamidedihydrochloride (KX2-391.HCl) was synthesized in four linear steps. Thefluoride of 4-(2-(4-(6-fluoropyridin-3-yl)phenoxy)ethyl)morpholine (5)was displaced by the anion of acetonitrile formed using commerciallyavailable NaHMDS. Acetonitrile was added slowly to a cooled mixture ofcompound 5 and base to form2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)acetonitrile (6). Inanother aspect of the invention, intermediate 5 may have a leaving groupother than fluorine. Thus, compounds of the general formula:

would be pursued where LG includes other leaving groups known to theskilled chemist.

Acid catalyzed methanolysis of2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)acetonitrile (6) wascarried out using a mixture of concentrated sulfuric and fuming sulfuricacid. The use of fuming sulfuric acid removed residual water from thereaction mixture and reduced the amount of carboxylic acid by-productformed. The reaction mixture was quenched by adding the reaction mixtureto a solution of saturated sodium bicarbonate and dichloromethane whilemaintaining the temperature below 20° C. Any carboxylic acid contaminantwas readily removed with aqueous work-up. In another aspect of theinvention, other acid catalyzed conditions are used by the skilledartisan for alcoholysis of the nitrile of compound 6 to produce compound7.

The resulting methyl2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)acetate (7) and benzylamine were coupled in anisole at high temperature to afford2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamide(KX2-391). An HCl solution formed by adding acetyl chloride to absoluteethanol was added to KX2-391 to form the bis-HCl salt,2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamidedihydrochloride, (KX2-di-HCl).

The synthesis of the mesylate salt of KX2-391 (KX2-391.MSA) is depictedin the scheme below:

2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamidemesylate (KX2-391.MSA) was synthesized in four linear steps startingfrom compound 5. The first three steps were carried out similar to theprocedure discussed above for KX2-391.2HCl to afford methyl2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)acetate (KX2-391).KX2-391 was converted to the methanesulfonate salt by treatment withmethanesulfonic acid (MSA) in acetone at 50° C. to afford2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamidemesylate (KX2-391.MSA).

In another aspect of the invention, intermediate 7 can be synthesizedhaving a group other than —C(O)OMe. The skilled chemist would pursueintermediate compounds of the general formula:

wherein the group “R” includes but is not limited to hydrogen and alkyl.

In one aspect, the invention relates to a process for preparing2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamidecomprising the steps of:

reacting 4-(2-chloroethyl)morpholine with 4-bromophenol to yield4-(2-(4-bromophenoxy)ethyl)morpholine; (2) coupling4-(2-(4-bromophenoxy)ethyl)morpholine with6-fluoropyridin-3-yl-3-boronic acid to yield4-(2-(4-(6-fluoropyridin-3-yl)phenoxy)ethyl)morpholine;

reacting 4-(2-(4-(6-fluoropyridin-3-yl)phenoxy)ethyl)morpholine withacetonitrile to yield2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)acetonitrile; (4)converting 2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)acetonitrileto methyl 2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)acetate; and(5) reacting methyl2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)acetate with benzylamineto yield2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamide.

In another aspect the invention relates to a process for preparing2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamidemesylate comprising the steps of: (1) reacting4-(2-chloroethyl)morpholine with 4-bromophenol to yield4-(2-(4-bromophenoxy)ethyl)morpholine; (2) coupling4-(2-(4-bromophenoxy)ethyl)morpholine with6-fluoropyridin-3-yl-3-boronic acid to yield4-(2-(4-(6-fluoropyridin-3-yl)phenoxy)ethyl)morpholine; (3) reacting4-(2-(4-(6-fluoropyridin-3-yl)phenoxy)ethyl)morpholine with acetonitrileto yield 2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)acetonitrile;(4) converting2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)acetonitrile to methyl2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)acetate; (5) reactingmethyl 2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)acetate withbenzylamine to yield2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamide; and(6) contacting2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamide withmethane sulfonic acid to yield2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamidemesylate.

In another aspect, the invention relates to a process for preparing2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamidemesylate comprising the step of contacting2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamide withmethane sulfonic acid to yield2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamidemesylate.

In another aspect, the invention relates to a process for preparing2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamidedihydrochloride comprising the steps of:

-   (1) reacting 4-(2-chloroethyl)morpholine with 4-bromophenol to yield    4-(2-(4-bromophenoxy)ethyl)morpholine;-   (2) coupling 4-(2-(4-bromophenoxy)ethyl)morpholine with    6-fluoropyridin-3-yl-3-boronic acid to yield    4-(2-(4-(6-fluoropyridin-3-yl)phenoxy)ethyl)morpholine;-   (3) reacting 4-(2-(4-(6-fluoropyridin-3-yl)phenoxy)ethyl)morpholine    with acetonitrile to yield    2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)acetonitrile;-   (4) converting    2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)acetonitrile to    methyl 2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)acetate;-   (5) reacting methyl    2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)acetate with    benzylamine to yield    2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamide;    and-   (6) contacting    2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamide    with hydrochloric acid to yield    2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamide    dihydrochloride.

In another aspect, the invention relates to a process for preparing2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamidedihydrochloride comprising the step of contacting2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamide withhydrochloric acid to yield2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamidedihydrochloride.

In another aspect, the invention relates to a process for preparing2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamidecomprising the step of reacting methyl2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)acetate with benzylamineto yield2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamide.

In another aspect, the invention relates to the process for preparing2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamidecomprising the steps of converting2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)acetonitrile to methyl2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)acetate; and reactingmethyl 2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)acetate withbenzylamine to yield2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamide.

In another aspect, the invention relates to the process for preparing2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamidecomprising the steps of reacting4-(2-(4-(6-fluoropyridin-3-yl)phenoxy)ethyl)morpholine with acetonitrileto yield 2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)acetonitrile;converting 2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)acetonitrileto methyl 2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)acetate; andreacting methyl 2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)acetatewith benzylamine to yield2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamide.

In another aspect, the invention relates to the process for preparing2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamidecomprising the steps of coupling 4-(2-(4-bromophenoxy)ethyl)morpholinewith 6-fluoropyridin-3-yl-3-boronic acid to yield4-(2-(4-(6-fluoropyridin-3-yl)phenoxy)ethyl)morpholine; reacting4-(2-(4-(6-fluoropyridin-3-yl)phenoxy)ethyl)morpholine with acetonitrileto yield 2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)acetonitrile;converting 2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)acetonitrileto methyl 2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)acetate; andreacting methyl 2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)acetatewith benzylamine to yield2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamide.

In another aspect, the invention relates to a process for preparing2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamidecomprising the step of reacting 4-(2-chloroethyl)morpholine with4-bromophenol to yield 4-(2-(4-bromophenoxy)ethyl)morpholine.

In another aspect, the invention relates to a process for preparing2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamidecomprising the step of coupling 4-(2-(4-bromophenoxy)ethyl)morpholinewith 6-fluoropyridin-3-yl-3-boronic acid to yield4-(2-(4-(6-fluoropyridin-3-yl)phenoxy)ethyl)morpholine.

In another aspect, the invention relates to a process for preparing2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamidecomprising the step of reacting4-(2-(4-(6-fluoropyridin-3-yl)phenoxy)ethyl)morpholine with acetonitrileto yield 2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)acetonitrile.

In another aspect, the invention relates to a process for preparing2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamidecomprising the steps of converting2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)acetonitrile to methyl2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)acetate.

In another aspect, the invention relates to the process described abovefor KX2-391 for preparing2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamidemesylate comprising the step of: contacting2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamide withmethane sulfonic acid to yield2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamidemesylate.

In another aspect, the invention relates to the process described abovefor KX2-391 for preparing2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamidedihydrochloride comprising the step of contacting2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamide withhydrochloric acid to yield2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamidedihydrochloride.

Compositions

The invention relates to substantially pure2-(5-(4-(2-morpholinoethoxy)phenyl)pyridine-2-yl)-N-benzylacetamide(KX2-391), and salts, solvates, hydrates, or prodrugs thereof:

Other names for the compound KX2-391 include2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamide andKX2-391 free base.

The invention relates to compositions and processes for the synthesis ofhighly purified KX2-391 (>98.0% as determined by HPLC) which is safe andsimple and which produces KX2-391 on a large scale (>100 g). Preferablythe synthesis produces the compound in high yield (>80%) and withlimited impurities.

In preferred embodiments, KX2-391 in the compositions of the instantinvention has a purity of greater than 98%. For example, the purity ofKX2-391 in the compositions of the invention is 98.5%, 99.0%, 99.5%,99.6%, 99.7%, 99.8% or 99.9%.

In preferred embodiments, the compositions and formulations of theinvention contain less than 2% impurities. For example, the compositionsand formulations of the invention contain less than 2% of any one of thefollowing impurities, or combinations thereof: ethyl chloride, ethanol,ethyl acetate, heptane, anisole, and palladium.

Some impurities are measured in parts per million, which is a relativeweight measurement equal to weight of solute/weight of solution X1,000,000, for example, the weight of ethyl chloride/weight of KX2-391di-HCl sample X 1,000,000; for example, the weight of ethylchloride/weight of KX2-391 mesylate sample X 1,000,000.

In other preferred embodiments the composition contains less than 250ppm ethyl chloride as determined by headspace gas chromatographyresidual solvent analysis. In an embodiment, the compounds andformulations of the present invention contain ethyl chloride in a rangefrom about 0 ppm to about 250 ppm (or any value within said range). Forexample, the compositions contain less than 200 ppm, less than 200 ppm,less than 150 ppm, less than 100 ppm, or less than 50 ppm ethylchloride.

The compounds and formulations of the present invention contain lessthan about 100 ppm palladium. In an embodiment, the compounds andformulations of the present invention contain palladium in a range fromabout 0 ppm to about 100 ppm (or any value within said range). Forexample, the compositions contain less than 75 ppm, less than 50 ppm,less than 30 ppm, less than 20 ppm, less than 10 ppm, or less than 5 ppmpalladium.

In an embodiment, the compounds and formulations of the presentinvention contain ethanol in a range from about 0 ppm to about 5000 ppm(or any value within said range). For example, the compositions containless than 4500 ppm, less than 4000 ppm, less than 3500 ppm, less than3000 ppm, less than 2500 ppm, or less than 2000 ppm ethanol.

In an embodiment, the compounds and formulations of the presentinvention contain ethyl acetate in a range from about 0 ppm to about50,000 ppm (or any value within said range). For example, thecompositions contain less than 48,000 ppm, less than 45,000 ppm, lessthan 40,000 ppm, less than 35,000 ppm, less than 30,000 ppm, or lessthan 25,000 ppm ethyl acetate.

In an embodiment, the compounds and formulations of the presentinvention contain heptane in a range from about 0 ppm to about 7,500 ppm(or any value within said range). For example, the compositions containless than 7,000 ppm, less than 6,500 ppm, less than 6,000 ppm, less than5,000 ppm, less than 3,000 ppm, or less than 1,000 ppm heptane.

In an embodiment, the compounds and formulations of the presentinvention contain anisole in a range from about 0 ppm to about 100 ppm(or any value within said range). For example, the compositions containless than 80 ppm, less than 75 ppm, less than 50 ppm, less than 25 ppm,less than 10 ppm, or less than 5 ppm anisole.

The invention relates to a composition that includes a substantiallypure solvate of KX2-391.

The invention also relates to a composition that includes asubstantially pure hydrate of KX2-391.

The invention also includes a substantially pure acid addition salt ofKX2-391. For example, a hydrochloride salt. The acid addition salt canbe, for example, a dihydrochloride salt. For example, the acid additionsalt can be a mesylate salt.

The invention relates to a composition that includes a substantiallypure acid addition salt of KX2-391.

The invention relates to a composition that includes a substantiallypure hydrochloride salt KX2-391. The invention relates to a compositionthat includes a substantially pure dihydrochloride salt of KX2-391.

The invention relates to a composition that includes a substantiallypure mesylate salt of KX2-391.

The invention also includes a prodrug of KX2-391.

The invention also includes a substantially pure, pharmaceuticallyacceptable salt of KX2-391.

The invention also relates to a composition that includes substantiallypure KX2-391 or a solvate, hydrate, or salt thereof, and at least onepharmaceutically acceptable excipient.

The invention relates to substantially pure2-(5-(4-(2-morpholinoethoxy)phenyl)pyridine-2-yl)-N-benzylacetamidedihydrochloride:

The invention relates to compositions and processes for the synthesis ofhighly purified KX2-391.2HCl or KX2-391.MSA (>98.0% as determined byHPLC) which is safe and simple and which produces KX2-391.2HCl orKX2-391.MSA respectively, on a large scale (>100 g) in high yield (>80%)and with limited ethyl chloride (<250 ppm ethyl chloride as determinedby headspace gas chromatography residual solvent analysis).

In preferred embodiments, KX2-391.2HCl in the compositions of theinstant invention has a purity of greater than 98%. For example, thepurity of KX2-391.2HCl in the compositions of the invention is 98.5%,99.0%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9%.

In preferred embodiments, the compositions and formulations of theinvention contain less than 2% impurities. For example, the compositionsand formulations of the invention contain less than 2% of any one of thefollowing impurities, or combinations thereof: ethyl chloride, ethanol,ethyl acetate, heptane, anisole, and palladium.

In other preferred embodiments the composition contains less than 250ppm ethyl chloride as determined by headspace gas chromatographyresidual solvent analysis. In an embodiment, the compounds andformulations of the present invention contain ethyl chloride in a rangefrom about 0 ppm to about 250 ppm (or any value within said range). Forexample, the compositions contain less than 200 ppm, less than 200 ppm,less than 150 ppm, less than 100 ppm, or less than 50 ppm ethylchloride.

The compounds and formulations of the present invention contain lessthan about 100 ppm palladium. In an embodiment, the compounds andformulations of the present invention contain palladium in a range fromabout 0 ppm to about 100 ppm (or any value within said range). Forexample, the compositions contain less than 75 ppm, less than 50 ppm,less than 30 ppm, less than 20 ppm, less than 10 ppm, or less than 5 ppmpalladium.

The invention also relates to a composition that includes substantiallypure KX2-391.2HCl and at least one pharmaceutically acceptableexcipient.

The invention relates to substantially pure2-(5-(4-(2-morpholinoethoxy)phenyl)pyridine-2-yl)-N-benzylacetamidemesylate (KX2-391.MSA):

The invention relates to compositions and processes for the synthesis ofhighly purified KX2-391.MSA (>98.0% as determined by HPLC) which is safeand simple and which produces KX2-391.MSA on a large scale (>100 g) inhigh yield (>80%) and with limited ethyl chloride (<250 ppm ethylchloride as determined by headspace gas chromatography residual solventanalysis).

In preferred embodiments, KX2-391.MSA in the compositions of the instantinvention has a purity of greater than 98%. For example, the purity ofKX2-391.MSA in the compositions of the invention is 98.5%, 99.0%, 99.5%,99.6%, 99.7%, 99.8% or 99.9%.

In preferred embodiments, the compositions and formulations of theinvention contain less than 2% impurities. For example, the compositionsand formulations of the invention contain less than 2% of any one of thefollowing impurities, or combinations thereof: ethyl chloride, ethanol,ethyl acetate, heptane, anisole, and palladium.

In other preferred embodiments the composition contains less than 250ppm ethyl chloride as determined by headspace gas chromatographyresidual solvent analysis. In an embodiment, the compounds andformulations of the present invention contain ethyl chloride in a rangefrom about 0 ppm to about 250 ppm (or any value within said range). Forexample, the compositions contain less than 200 ppm, less than 200 ppm,less than 150 ppm, less than 100 ppm, or less than 50 ppm ethylchloride.

The compounds and formulations of the present invention contain lessthan about 100 ppm palladium. In an embodiment, the compounds andformulations of the present invention contain palladium in a range fromabout 0 ppm to about 100 ppm (or any value within said range). Forexample, the compositions contain less than 75 ppm, less than 50 ppm,less than 30 ppm, less than 20 ppm, less than 10 ppm, or less than 5 ppmpalladium.

The invention also relates to a composition that includes substantiallypure KX2-391.MSA and at least one pharmaceutically acceptable excipient.

Certain compounds of the invention are non-ATP competitive kinaseinhibitors.

For example, the compounds of the invention are useful to treat orprevent a microbial infection, such as a bacterial, fungal, parasitic orviral infection.

Certain pharmaceutical compositions of the invention includesubstantially pure KX2-391.2HCl.

A compound of the invention may be used as a pharmaceutical agent. Forexample, a compound of the invention is used as an anti-proliferativeagent, for treating humans and/or animals, such as for treating humansand/or other mammals. The compounds may be used without limitation, forexample, as anti-cancer, anti-angiogenesis, anti-microbial,anti-bacterial, anti-fungal, anti-parasitic and/or anti-viral agents.Additionally, the compounds may be used for other cellproliferation-related disorders such as diabetic retinopathy, maculardegeneration and psoriases. Anti-cancer agents include anti-metastaticagents.

The compound of the invention used as a pharmaceutical agent may be, forexample, substantially pure KX2-391, KX2-391.2HCl, or KX2-391.MSA.

The present invention provides compositions and formulations whichcontain limited impurities. The compounds and formulations of thepresent invention have a purity greater than about 98.0% as determinedby known methods in the art, for example, HPLC. In an embodiment, thecompounds and formulations of the present invention have a purityranging from about 99.0% to about 100% (or any value within said range).For example, such compounds, compositions, or formulations can have apurity of 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%,99.0%, 99.1%, 99.2%, 99.3, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%.

In order to elicit the maximum pharmacodynamic and therapeutic effect ofthe compositions and formulations of the present invention, it isbeneficial to limit the levels of impurities such as ethyl chloride andpalladium. These impurities can result in undesirable toxicity.

In preferred embodiments, the compositions and formulations of theinvention contain less than 2% impurities. For example, the compositionsand formulations of the invention contain less than 2% of any one of thefollowing impurities, or combinations thereof: ethyl chloride, ethanol,ethyl acetate, heptane, anisole, and palladium.

In other preferred embodiments the composition contains less than 250ppm ethyl chloride as determined by headspace gas chromatographyresidual solvent analysis. In an embodiment, the compounds andformulations of the present invention contain ethyl chloride in a rangefrom about 0 ppm to about 250 ppm (or any value within said range). Forexample, the compositions contain less than 200 ppm, less than 200 ppm,less than 150 ppm, less than 100 ppm, or less than 50 ppm ethylchloride.

The compounds and formulations of the present invention contain lessthan about 100 ppm palladium. In an embodiment, the compounds andformulations of the present invention contain palladium in a range fromabout 0 ppm to about 100 ppm (or any value within said range). Forexample, the compositions contain less than 75 ppm, less than 50 ppm,less than 30 ppm, less than 20 ppm, less than 10 ppm, or less than 5 ppmpalladium.

Methods of Use

Because kinases are involved in the regulation of a wide variety ofnormal cellular signal transduction pathways (e.g., cell growth,differentiation, survival, adhesion, migration, etc.), kinases arethought to play a role in a variety of diseases and disorders. Thus,modulation of kinase signaling cascades may be an important way to treator prevent such diseases and disorders. Such diseases and disordersinclude, for example, cancers, osteoporosis, cardiovascular disorders,immune system dysfunction, type II diabetes, obesity, and transplantrejection.

Compounds of the invention are useful in modulation a component of thekinase signaling cascade. Some compounds may be useful in modulation ofmore than one component of a kinase signaling cascade. The phrase“modulates one or more components of a protein kinase signaling cascade”means that one or more components of the kinase signaling cascade areaffected such that the functioning of a cell changes. Components of aprotein kinase signaling cascade include any proteins involved directlyor indirectly in the kinase signaling pathway including secondmessengers and upstream and downstream targets.

A number of protein kinases and phosphatases are known, and are targetsfor the development of therapeutics. See, e.g., Hidaka and Kobayashi,Annu. Rev. Pharmacol. Toxicol, 1992, 32:377-397; Davies et al., Biochem.J., 2000, 351:95-105, each of which is incorporated by reference herein.

One family of kinases, the protein tyrosine kinases are divided into twolarge families: receptor tyrosine kinases, or RTKs (e.g., insulinreceptor kinase (IRK), epidermal growth factor receptor (EGFR), basicfibroblast growth factor receptor (FGFR), platelet-derived growth factorreceptor (PDGFR), vascular endothelial growth factor receptor (VEGFR-2or Flk1/KDR), and nerve growth factor receptor (NGFR)) and nonreceptortyrosine kinases, or NRTKs (e.g., the Src family (Src, Fyn, Yes, Blk,Yrk, Fgr, Hck, Lck, and Lyn), Fak, Jak, Abl and Zap70). See, forexample, Parang and Sun, Expert Opin. Ther. Patents, 2005, 15:1183-1207,incorporated by reference herein.

Because of the role of Src kinases in a variety of cancers, thesekinases are the subject of a number of studies relating to thedevelopment of Src inhibitors as cancer therapeutics, including highlymetastatic cancer cell growth. Src inhibitors are sought as therapeuticsfor a variety of cancers, including, for example, colon cancer,precancerous colon lesions, ovarian cancer, breast cancer, epithelialcancers, esophageal cancer, non-small cell lung cancer, pancreaticcancer, and others. See, e.g., Frame, Biochim. Biophys. Acta, 2002,1602:114-130 and Parang and Sun, Expert Opin. Ther. Patents, 2005,15:1183-1207.

Inhibition of other kinases may be useful in the treatment andmodulation of other types of diseases and disorders. For example,various eye diseases may be inhibited or prevented by administration ofVEGF receptor tyrosine kinase inhibitors. Inhibitors of the tyrosinephosphatase PTP-1B and/or glycogen phosphorylase may provide treatmentsfor Type II diabetes or obesity. Inhibitors of p56lck may be useful intreating immune system disorders. Other targets include HIV reversetranscriptase, thromboxane synthase, EGFRTK, p55 fyn, etc.

Compounds of the invention may be Src signaling inhibitors that bind inthe Src peptide substrate site. The activity of various compounds of theinvention has been studied in c-Src (527F, constitutively active andtransforming) transformed NIH3T3 cells and in human colon cancer cells(HT29). For example, in these cell lines, KX2-391 was shown to reducethe phosphorylation level of known Src protein substrates in adose-dependent fashion and in good correlation with growth inhibitoryeffects. Thus, in some embodiments, compounds of the invention maydirectly inhibit Src, and may do so by binding in the peptide bindingsite (as opposed to binding at an allosteric site).

Molecular modeling experiments have been performed which show thatcompounds of the invention fit into the model Src substrate site (See,e.g., U.S. Pat. Nos. 7,005,445 and 7,070,936). Modeling is also used toretool the Src kinase inhibitor scaffolds in order to target otherkinases, simply by using a different set of side chains present on themolecules and/or modifying the scaffold itself.

Without wishing to be bound by theory, it is believed that theconformation of some kinases (e.g., Src) outside cells relative to theconformation inside cells is markedly different, because inside cells,many kinases are is embedded in multiprotein signaling complexes. Thus,because the peptide substrate binding site is not well formed in anisolated kinase (as shown by Src x-ray structures), it is believed thatthe activity against isolated kinase for a peptide substrate bindinginhibitor would be weak. Binding to this site in an isolated kinaseassay requires the inhibitor to capture the very small percentage oftotal protein in an isolated enzyme assay that is in the sameconformation that exists inside cells. This requires a large excess ofthe inhibitor to drain significant amounts of the enzyme from thecatalytic cycle in the assay in order to be detectable.

However, for cell-based assays, a large inhibitor excess is not neededbecause the peptide binding site is expected to be formed. In cell-basedSrc assays, SH2 & SH3 domain binding proteins have already shifted theSrc conformation so that the peptide substrate binding site is fullyformed. Thus, low concentrations of the inhibitor can remove the enzymefrom the catalytic cycle since all of the enzyme is in the tight bindingconformation.

The vast majority of known kinase inhibitors are ATP competitive andshow poor selectivity in a panel of isolated kinase assays. However,many of the compounds of the invention are thought to be peptidesubstrate binding inhibitors. Thus, traditional high throughputscreening of compounds against isolated enzymes, such as Src, would notresult in the discovery of compounds of the invention.

Compounds of the invention may be a kinase inhibitor. The compound ofthe invention may be a non-ATP competitive kinase inhibitor. Thecompound of the invention may inhibit a kinase directly, or it mayaffect the kinase pathway. In one embodiment, the compound inhibits oneor more components of a protein kinase signaling cascade. In anotherembodiment, the compound is an allosteric inhibitor. In anotherembodiment, the compound is a peptide substrate inhibitor. In anotherembodiment, the compound does not inhibit ATP binding to a proteinkinase. In one embodiment, the compound inhibits a Src family proteinkinase. In another embodiment, the Src family protein kinase is pp60^(c-src) tyrosine kinase.

The compounds of the present invention are useful as pharmaceuticalagents, for example, as therapeutic agents for treating humans andanimals. The compounds may be used without limitation, for example, asanti-cancer, anti-angiogenesis, anti-metastatic, anti-microbial,anti-bacterial, anti-fungal, anti-parasitic and/or anti-viral agents.

In one embodiment, the administration of the compound is carried outorally, parentally, subcutaneously, intravenously, intramuscularly,intraperitoneally, by intranasal instillation, by intracavitary orintravesical instillation, topically e.g., by administering drops intothe car, intraarterially, intralesionally, by metering pump, or byapplication to mucous membranes. In another embodiment, the compound isadministered with a pharmaceutically acceptable carrier.

Cancer

There is considerable recent literature support for targeting pp 60c-src(Src) as a broadly useful approach to cancer therapy without resultingin serious toxicity. For example, tumors that display enhanced EGFreceptor PTK signaling, or overexpress the related Her-2/neu receptor,have constitutively activated Src and enhanced tumor invasiveness.Inhibition of Src in these cells induces growth arrest, triggersapoptosis, and reverses the transformed phenotype (Karni et al. (1999)Oncogene 18(33): 4654-4662). It is known that abnormally elevated Srcactivity allows transformed cells to grow in an anchorage-independentfashion. This is apparently caused by the fact that extracellular matrixsignaling elevates Src activity in the FAK/Src pathway, in a coordinatedfashion with mitogenic signaling, and thereby blocks an apoptoticmechanism which would normally have been activated. Consequently FAK/Srcinhibition in tumor cells may induce apoptosis because the apoptoticmechanism which would have normally become activated upon breaking freefrom the extracellular matrix would be induced (Hisano, et al., Proc.Annu. Meet. Am. Assoc. Cancer Res. 38:A1925 (1997)). Additionally,reduced VEGF mRNA expression was noted upon Src inhibition and tumorsderived from these Src-inhibited cell lines showed reduced angiogenicdevelopment (Ellis et al., Journal of Biological Chemistry 273(2):1052-1057 (1998)).

Src has been proposed to be a “universal” target for cancer therapysince it has been found to be overactivated in a growing number of humantumors (Levitzki, Current Opinion in Cell Biology, 8, 239-244 (1996);Levitzki, Anti-Cancer Drug Design, 11, 175-182 (1996)). The potentialbenefits of Src inhibition for cancer therapy appear to be four-foldinhibition of uncontrolled cell growth caused by autocrine growth factorloop effects, inhibition of metastasis due to triggering apoptosis uponbreaking free from the cell matrix, inhibition of tumor angiogenesis viareduced VEGF levels, and low toxicity.

Prostate cancer cells have been reported to have both an over expressionof paxillin and p130cas and are hyperphosphorylated (Tremblay et al.,Int. J. Cancer, 68, 164-171, 1996) and may thus be a prime target forSrc inhibitors.

The invention includes a method of preventing or treating a cellproliferation disorder by administering a pharmaceutical compositionthat includes a substantially pure KX2-391, or a salt, solvate, hydrate,or prodrug thereof, and at least one pharmaceutically acceptableexcipient to a subject in need thereof. The invention includessubstantially pure KX2-391 bis-HCl. The invention includes substantiallypure KX2-391 mesylate.

For example, the cell proliferation disorder is pre-cancer or cancer.The cell proliferation disorder treated or prevented by the compounds ofthe invention may be a cancer, such as, for example, colon cancer orlung cancer. The cell proliferation disorder treated or prevented by thecompounds of the invention may be a hyperproliferative disorder. Thecell proliferation disorder treated or prevented by the compounds of theinvention may be psoriases.

Treatment or prevention of the proliferative disorder may occur throughthe inhibition of a tyrosine kinase. For example, the tyrosine kinasecan be a Src kinase or focal adhesion kinase (FAK).

The invention is also drawn to a method of treating or preventing canceror a proliferation disorder in a subject, comprising administering acomposition comprising an effective amount of a substantially pureKX2-391, or a salt, solvate, hydrate, or prodrug thereof, for example,substantially pure KX2-391, KX2-391.2HCl or KX2-391.MSA.

Hearing Loss

As described herein, a compound of the invention may be used to protectagainst or prevent hearing loss in a subject. In order to protectagainst hearing loss, the compound may be administered prior to noiseexposure or exposure to a drug which induces hearing loss to preventhearing loss or to reduce the level of hearing loss. Such drugs whichinduce hearing loss may include chemotherapeutic drugs (e.g.,platinum-based drugs which target hair cells) and aminoglycosideantibiotics. A compound of the invention may provide a synergisticeffect with certain cancer drugs. For example, promising inhibitors canbe screened in primary human tumor tissue assays, particularly to lookfor synergy with other known anti-cancer drugs. In addition, the proteinkinase inhibitors may reduce toxicity of certain cancer drugs (e.g.,platinum-based drugs which are toxic to the cochlea and kidney), therebyallowing increased dosage.

Alternatively, a compound of the invention may be used to treat hearingloss in a subject. In this embodiment, the compound is administered tothe subject subsequent to the initiation of hearing loss to reduce thelevel of hearing loss. A compound of the invention may be involved inmodulating a kinase cascade, e.g. a kinase inhibitor, a non-ATPcompetitive inhibitor, a tyrosine kinase inhibitor, a Src inhibitor or afocal adhesion kinase (FAK) modulator. Although not wishing to be boundby theory, it is believed that the administration of kinase inhibitorsprevents apoptosis of cochlear hair cells, thereby preventing hearingloss. In one embodiment, administration of a compound of the inventionis administered to a subject suffering from hearing loss in order toprevent further hearing loss. In another embodiment, administration of acompound of the invention is administered to a subject suffering fromhearing loss in order to restore lost hearing. In particular, followingnoise exposure, the tight cell junctures between the cochlear haircells, as well as the cell-extracellular matrix interaction, are tornand stressed. The stressing of these tight cell junctures initiatesapoptosis in the cells through a complex signaling pathway in whichtyrosine kinases act as molecular switches, interacting with focaladhesion kinase to transduce signals of cell-matrix disruptions to thenucleus. It is believed that the administration of kinase inhibitorsprevents the initiation of apoptosis in this cascade.

The identification of apoptosis in the noise-exposed cochlea hasgenerated a number of new possibilities for the prevention ofnoise-induced hearing loss (NIHL) (Hu, et al.; 2000, Acta. Otolaryngol.,120, 19-24). For example, the ear can be protected from NIHL byadministration of antioxidant drugs to the round window of the ear(Hight, et al.; 2003, Hear. Res., 179, 21-32; Hu, et al.; Hear. Res.113, 198-206). Specifically, NIHL has been reduced by the administrationof FDA-approved antioxidant compounds (N-L-acetylcysteine (L-NAC) andsalicylate) in the chinchilla (Kopke, et al.; 2000, Hear. Res., 149,138-146). Moreover, Harris et al. have recently described prevention ofNIHL with Src-PTK inhibitors (Harris, et al.; 2005, Hear. Res., 208,14-25). Thus, it is hypothesized that the administration of a compoundof the instant invention which modulates the activity of kinases, isuseful for treating hearing loss.

Changes in cell attachment or cell stress can activate a variety ofsignals through the activation of integrins and through thephosphorylation of PTKs, including the Src family of tyrosine kinases.Src interactions have been linked to signaling pathways that modify thecytoskeleton and activate a variety of protein kinase cascades thatregulate cell survival and gene transcription (reviewed in Giancotti andRuoslahti; 1999, Science, 285, 1028-1032). In fact, recent results haveindicated that outer hair cells (OHC), which had detached at the cellbase following an intense noise exposure, underwent apoptotic celldeath. Specifically, the Src PTK signaling cascade is thought to beinvolved in both metabolic- and mechanically-induced initiation ofapoptosis in sensory cells of the cochlea. In a recent study, Srcinhibitors provided protection from a 4 hour, 4 kHz octave band noise at106 dB, indicating that Src-PTKs might be activated in outer hair cellsFollowing noise exposure (Harris, et al.; 2005, Hear. Res., 208, 14-25).Thus, compounds of the instant invention that modulate the activity ofSrc, are useful in treating hearing loss.

Another aspect of the invention includes a method of protecting againstor treating hearing loss in a subject comprising administering acomposition comprising an effective amount of a substantially pureKX2-391, or a salt, solvate, hydrate, or prodrug thereof, for example,substantially pure KX2-391, KX2-391.2HCl, or KX2-391.MSA.

In one embodiment, the compound is administered before initiation ofhearing loss. In another embodiment, the compound is administered afterinitiation of hearing loss.

In one embodiment, the compound is administered in combination with adrug that causes hearing loss e.g., cis platinum or an aminoglycosideantibiotic. In another embodiment, the compound is administered incombination with a drug that targets hairy cells.

Osteoporosis

The present invention relates to a method for protecting against ortreating osteoporosis in a subject. This method involves administeringan effective amount of a compound of the invention to the subject toprotect against or to treat osteoporosis. In order to protect againstosteoporosis, the compound may be administered prior to the developmentof osteoporosis. Alternatively, the compound may be used to treatosteoporosis in a subject. In one embodiment, the compound isadministered to the subject subsequent to the initiation of osteoporosisto reduce the level of osteoporosis.

A compound of the invention can be, e.g. a non-ATP competitiveinhibitor. The compound of the invention can modulate a kinase signalingcascade, depending upon the particular side chains and scaffoldmodifications selected. The compound of the invention can be a kinaseinhibitor. For example, the compound can be a protein tyrosine kinase(PTK) inhibitor. The proline-rich tyrosine kinase (PYK2; also known ascell adhesion kinase β, related adhesion focal tyrosine kinase, orcalcium-dependent tyrosine kinase) and focal adhesion kinase (FAK) aremembers of a distinct family of non receptor protein-tyrosine kinasesthat are regulated by a variety of extracellular stimuli (Avraham, etal.; 2000, Cell Signal., 12, 123-133; Schlaepfer, et al.; 1999, Prog.Biophys. Mol. Biol., 71, 435-478). The compound of the invention can bea Src inhibitor. It has been shown that Src deficiency is associatedwith osteoporosis in mice, because of loss of osteoclast function(Soriano, et al.; 1991, Cell, 64, 693-702). Alternatively, the compoundof the invention can modulate the expression of interleukin-1 receptorassociated kinase M (IRAK-M). Mice that lack IRAK-M develop severeosteoporosis, which is associated with the accelerated differentiationof osteoclasts, an increase in the half-life of osteoclasts, and theiractivation (Hongmei, et al.; 2005, J. Exp. Med., 201, 1169-1177).

Multinucleated osteoclasts originate from the fusion of mononuclearphagocytes and play a major role in bone development and remodeling viathe resorption of bone. Osteoclasts are multinucleated, terminallydifferentiated cells that degrade mineralized matrix. In normal bonetissue, there is a balance between bone formation by osteoblasts andbone resorption by osteoclasts. When the balance of this dynamic andhighly regulated process is disrupted, bone resorption can exceed boneformation resulting in quantitative bone loss. Because osteoclasts areessential for the development and remodeling of bone, increases in theirnumber and/or activity lead to diseases that are associated withgeneralized bone loss (e.g., osteoporosis) and others with localizedbone loss (e.g., rheumatoid arthritis, periodontal disease).

Osteoclasts and osteoblasts both command a multitude of cellularsignaling pathways involving protein kinases. Osteoclast activation isinitiated by adhesion to bone, cytoskeletal rearrangement, formation ofthe sealing zone, and formation of the polarized ruffled membrane. It isbelieved that protein-tyrosine kinase 2 (PYK2) participates in thetransfer of signals from the cell surface to the cytoskeleton, as it istyrosine phosphorylated and activated by adhesion-initiated signaling inosteoclasts (Duong, et al.; 1998, J. Clin. Invest., 102, 881-892).Recent evidence has indicated that the reduction of PYK2 protein levelsresults in the inhibition of osteoclast formation and bone resorption invitro (Duong, et al.; 2001, J. Bio. Chem., 276, 7484-7492). Therefore,the inhibition of PYK2 or other protein tyrosine kinases might reducethe level of osteoporosis by decreasing osteoclast formation and boneresorption. Thus, without wishing to be bound by theory, it ishypothesized that the administration of a compound of the instantinvention will modulate kinase (e.g. PTK) activity and therefore resultin the inhibition of osteoclast formation and/or bone resorption,thereby treating osteoporosis.

Src tyrosine kinase stands out as a promising therapeutic target forbone disease as validated by Src knockout mouse studies and in vitrocellular experiments, suggesting a regulatory role for Src in bothosteoclasts (positive) and osteoblasts (negative). In osteoclasts, Srcplays key roles in motility, polarization, survival, activation (ruffledborder formation) and adhesion, by mediating various signal transductionpathways, especially in cytokine and integrin signaling (Parang and Sun;2005, Expert Opin. Ther. Patents, 15, 1183-1207). Moreover, targeteddisruption of the src gene in mice induces osteopetrosis, a disordercharacterized by decreased bone resorption, without showing any obviousmorphological or functional abnormalities in other tissues or cells(Soriano, et al.; 1991, Cell, 64, 693-702). The osteopetrotic phenotypeof src^(−/−) mice is cell-autonomous and results from defects in matureosteoclasts, which normally express high levels of Src protein (Home, etal.; 1991, Cell, 119, 1003-1013). By limiting the effectiveness of Srctyrosine kinase, which triggers osteoclast activity and inhibitsosteoblasts, Src inhibitors are thought to lessen bone break down andencourage bone formation. Because osteoclasts normally express highlevels of Src, inhibition of Src kinase activity might be useful in thetreatment of osteoporosis (Missbach, et al.; 1999, Bone, 24, 437-449).Thus, the PTK inhibitors of the instant invention that modulate theactivity of Src, are useful in treating osteoporosis.

For example, a knock-out of the Src gene in mice led to only one defect,namely osteoclasts that fail to form ruffled borders and consequently donot resorb bone. However, the osteoclast bone resorb function wasrescued in these mice by inserting a kinase defective Src gene(Schwartzberg et al., (1997) Genes & Development 11: 2835-2844). Thissuggested that Src kinase activity can be inhibited in vivo withouttriggering the only known toxicity because the presence of the Srcprotein is apparently sufficient to recruit and activate other PTKs(which are essential for maintaining osteoclast function) in anosteoclast essential signaling complex.

Another aspect of the invention includes a method of protecting againstor treating osteoporosis in a subject comprising administering acomposition comprising an effective amount of a substantially pureKX2-391, or a salt, solvate, hydrate, or prodrug thereof, for example,substantially pure KX2-391, KX2-391.2HCl, or KX2-391.MSA.

In one embodiment, the compound is administered before initiation ofosteoporosis. In another embodiment, the compound is administered afterinitiation of osteoporosis.

Obesity

As described herein, a compound of the invention may be used to protectagainst or prevent obesity in a subject. In order to protect againstobesity, the compound may be administered prior to the development ofobesity in a subject. For example, the compound may be administered toprevent or reduce weight gain. Alternatively, the compound may be usedto treat obesity in a subject. A compound of the instant invention maybe involved in modulating a kinase signaling cascade, e.g., a kinaseinhibitor, a non-ATP competitive inhibitor, a tyrosine kinase inhibitor,a protein tyrosine phosphatase inhibitor, or a protein-tyrosinephosphatase 1B inhibitor.

Obesity is often associated with diabetes and increased insulinresistance in insulin responsive tissues, such as skeletal muscle,liver, and white adipose tissue (Klaman, et al.; 2000, Mol. Cell. Biol.,20, 5479-5489). Insulin plays a critical role in the regulation ofglucose homeostasis, lipid metabolism, and energy balance. Insulinsignaling is initiated by binding of insulin to the insulin receptor(IR), a receptor tyrosine kinase. Insulin binding evokes a cascade ofphosphorylation events, beginning with the autophosphorylation of the IRon multiple tyrosyl residues. Autophosphorylation enhances IR kinaseactivity and triggers downstream signaling events. The stimulatoryeffects of protein tyrosine kinases and the inhibitory effects ofprotein tyrosine phosphatases largely define the action of insulin.Appropriate insulin signaling minimizes large fluctuations in bloodglucose concentrations and ensures adequate delivery of glucose tocells. Since insulin stimulation leads to multiple tyrosylphosphorylation events, enhanced activity of one or moreprotein-tyrosine phosphatases (PTPs) could lead to insulin resistance,which may lead to obesity. Indeed, increased PTP activity has beenreported in several insulin-resistant states, including obesity (Ahmad,et al.; 1997, Metabolism, 46, 1140-1145). Thus, without wishing to bebound by theory, the administration of a compound of the instantinvention modulates kinase (e.g., PTP) activity, thereby treatingobesity in a subject.

Insulin signaling begins with the activation of the IR via tyrosinephosphorylation and culminates in the uptake of glucose into cells bythe glucose transporter, GLUT4 (Saltiel and Kahn; 2001, Nature, 414,799-806). The activated IR must then be deactivated and returned to abasal state, a process that is believed to involve protein-tyrosinephosphatase-1B (PTP-1B) (Ahmad, et al; 1997, J. Biol. Chem., 270,20503-20508). Disruption of the gene that codes for PTP-1B in miceresults in sensitivity to insulin and increased resistance todiet-induced obesity (Elchebly, et al.; 1999, Science, 283, 1544-1548;Klaman, et al.; 2000, Mol. Cell. Biol., 20, 5479-5489). The decreasedadiposity in PTP-1B deficient mice was due to a marked reduction in fatcell mass without a decrease in adipocyte number (Klaman, et al.; 2000,Mol. Cell. Biol., 20, 5479-5489). Moreover, leanness in PTP-1B-deficientmice was accompanied by increased basal metabolic rate and total energyexpenditure, without marked alteration of uncoupling protein mRNAexpression. The disruption of the PTP-1B gene demonstrated that alteringthe activity of PTP-1B can modulate insulin signaling anddietary-induced obesity in vivo. Thus, without wishing to be bound bytheory, the administration of a compound of the instant invention thatmodulates insulin signaling (e.g., PTP-1B activity), is useful intreating obesity in a subject.

Another aspect of the invention includes a method of protecting againstor treating obesity in a subject comprising administering a compositioncomprising an effective amount of a substantially pure KX2-391, or asalt, solvate, hydrate, or prodrug thereof, for example, substantiallypure KX2-391, KX2-391.2HCl, or KX2-391.MSA.

In one embodiment, the compound is administered before the subject isobese. In another embodiment, the compound is administered after thesubject is obese.

Diabetes

As described herein, a compound of the invention may be used to protectagainst or prevent diabetes in a subject. In order to protect againstdiabetes, the compound may be administered prior to the development ofdiabetes in a subject. Alternatively, the compound may be used to treatdiabetes in a subject. The compound of the instant invention may beinvolved in modulating a kinase signaling cascade, e.g. a kinaseinhibitor, a non-ATP competitive inhibitor, a tyrosine kinase inhibitor,a phosphatase and tension homologue on chromosome 10 (PTEN) inhibitor,or a sequence homology 2-containing inositol 5′-phosphatase 2 (SHIP2)inhibitor.

Type 2 diabetes mellitus (T2DM) is a disorder of dysregulated energymetabolism. Energy metabolism is largely controlled by the hormoneinsulin, a potent anabolic agent that promotes the synthesis and storageof proteins, carbohydrates and lipids, and inhibits their breakdown andrelease back into the circulation. Insulin action is initiated bybinding to its tyrosine kinase receptor, which results inautophosphorylation and increased catalytic activity of the kinase(Patti, et al.; 1998, J. Basic Clin. Physiol. Pharmacol. 9, 89-109).Tyrosine phosphorylation causes insulin receptor substrate (IRS)proteins to interact with the p85 regulatory subunit ofphosphatidylinositol 3-kinase (PI3K), leading to the activation of theenzyme and its targeting to a specific subcellular location, dependingon the cell type. The enzyme generates the lipid productphosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P₃), whichregulates the localization and activity of numerous proteins (Kido, etal.; 2001, J. Clin. Endocrinol. Metab., 86, 972-979). PI3K has anessential role in insulin-stimulated glucose uptake and storage,inhibition of lipolysis and regulation of hepatic gene expression(Saltiel, et al.; 2001, Nature, 414, 799-806). Overexpression ofdominant-interfering forms of PI3K can block glucose uptake andtranslocation of glutamate transporter four, GLUT4, to the plasmamembrane (Quon, et al.; 1995, Mol. Cell. Biol., 15, 5403-5411). Thus,the administration of a compound of the instant invention that modulateskinase (e.g. PI3K) activity, and therefore results in increased glucoseuptake, is useful in treating diabetes. PTEN is a major regulator ofPI3K signaling in may cell types, and functions as a tumor suppressordue to antagonism of the anti-apoptotic, proliferative and hypertrophicactivities of the PI3K pathway (Goberdhan, et al.; 2003, Hum. Mol.Genet., 12, R239-R248; Leslie, et al.; 2004, J. Biochem., 382, 1-11).Although not wishing to be bound by theory, it is believed that PTENattenuates the PI3K pathway by dephosphorylation of the PtdIns(3,4,5)P₃molecule, degrading this important lipid second messenger toPtdIns(4,5)P₂. In a recent study, reduction of endogenous PTEN proteinby 50% using small interfering RNA (siRNA) enhanced insulin-dependentincreases in PtdIns(3,4,5)P₃ levels, and glucose uptake (Tang, et al.;2005, J Biol. Chem., 280, 22523-22529). Thus, without wishing to bebound by theory, it is hypothesized that the administration of acompound of the instant invention that modulates PTEN activity, andtherefore results in increased glucose uptake, is useful for treatingdiabetes.

PtdIns(3,4,5)P₃ levels are also controlled by the family of SRC homology2 (SH2)-containing inositol 5′-phosphatase (SHIP) proteins, SHIP1 andSHIP2 (Lazar and Saltiel; 2006, Nature Reviews, 5, 333-342). SHIP2,expressed in skeletal muscle, among other insulin-sensitive tissues,catalyzes the conversion of PtdIns(3,4,5)P₃ into PtdIns(3,4)P₂ (Pesesse,et al.; 1997; Biochem Biophys. Res. Commun., 239, 697-700; Backers, etal.; 2003, Adv. Enzyme Regul., 43, 15-28; Chi, et a; 2004, J. Biol.Chem., 279, 44987-44995; Sleeman, et al.; 2005, Nature Med., 11,199-205). Overexpression of SHIP2 markedly reduced insulin-stimulatedPtdIns(3,4,5)P₃ levels, consistent with the proposed capacity of SHIP2to attenuate the activation of downstream effectors of PI3K (Ishihara,et al.; 1999, Biochem. Biophys. Res. Commun., 260, 265-272). Thus,without wishing to be bound by theory, it is hypothesized that theadministration of a compound of the instant invention which modulatesSHIP2 activity, and therefore results in increased glucose uptake, isuseful for treating diabetes.

Another aspect of the invention includes a method of protecting againstor treating diabetes in a subject comprising administering a compositioncomprising an effective amount of a substantially pure KX2-391, or asalt, solvate, hydrate, or prodrug thereof, for example, substantiallypure KX2-391, KX2-391.2HCl, or KX2-391.MSA.

In one embodiment, the compound is administered before initiation of thediabetes. In another embodiment, the compound is administered afterinitiation of disease.

Ophthalmic Disease

As described herein, a compound of the invention may be used to protectagainst or prevent ophthalmic (eye) disease in a subject. In order toprotect against eye disease, the compound may be administered prior tothe development of eye disease in a subject. Alternatively, the compoundmay be used to treat eye disease in a subject, e.g. maculardegeneration, retinopathy, and macular edema. The compound of theinstant invention may be involved in modulating a kinase cascade, e.g. akinase inhibitor, a non-ATP competitive inhibitor, a tyrosine kinaseinhibitor, e.g. a vascular endothelial growth factor (VEGF) receptortyrosine kinase inhibitor.

Vision-threatening neovascularization of the physiologically avascularcornea can occur. The proliferative retinopathies, principally diabeticretinopathy and age-related macular degeneration, are characterized byincreased vascular permeability, leading to retinal edema and subretinalfluid accumulation, and the proliferation of new vessels that are proneto hemorrhage. Angiogenesis, the formation of new blood vessels frompreexisting capillaries, is an integral part of both normal developmentand numerous pathological processes. VEGF, a central mediator of thecomplex cascade of angiogenesis and a potent permeability factor, is anattractive target for novel therapeutics. VEGF is the ligand for twomembrane-bound tyrosine kinase receptors, VEGFR-1 and VEGFR-2. Ligandbinding triggers VEGFR dimerization and transphosphorylation withsubsequent activation of an intracellular tyrosine kinase domain. Theensuing intracellular signaling axis results in vascular endothelialcell proliferation, migration, and survival. Thus, without wishing to bebound by theory, it is hypothesized that the administration of acompound of the instant invention which modulates kinase activity, e.g.tyrosine kinase activity, and results in the inhibition of angiogenesisand/or neovascularization, is useful for treating an eye disease, e.g.macular degeneration, retinopathy and/or macular edema.

Macular degeneration is characterized by VEGF-mediated retinal leakage(an increase in vascular permeability) and by the abnormal growth ofsmall blood vessels in the back of the eye (angiogenesis). VEGF has beenidentified in neovascular membranes in both diabetic retinopathy andage-related macular degeneration, and intraocular levels of the factorcorrelate with the severity of neovascularization in diabeticretinopathy (Kvanta, et al.; 1996, Invest. Ophthal. Vis. Sci., 37,1929-1934.; Aiello et al., 1994, N. Engl. J. Med., 331, 1480-1487).Therapeutic antagonism of VEGF in these models results in significantinhibition of both retinal and choroidal neovascularization, as well asa reduction in vascular permeability (Aiello, et al.; 1995, Proc. Natl.Acad. Sci. USA., 92, 10457-10461; Krzystolik, et al.; 2002, Arch.Ophthal., 120, 338-346; Qaum, et al.; 2001, Invest. Ophthal. Vis. Sci.,42, 2408-2413). Thus, without wishing to be bound by theory, it ishypothesized that the administration of a compound of the instantinvention which modulates VEGF activity, and results in the inhibitionof angiogenesis and/or neovascularization, is useful for treating an eyedisease, e.g. macular degeneration, retinopathy and/or macular edema.

Another aspect of the invention includes a method of protecting againstor treating ophthalmic diseases e.g., macular degeneration, retinopathy,macular edema, etc. in a subject comprising administering a compositioncomprising an effective amount of a substantially pure KX2-391, or asalt, solvate, hydrate, or prodrug thereof, for example, substantiallypure KX2-391, KX2-391.2HCl, or KX2-391.MSA.

In one embodiment, the compound is administered before initiation of theophthalmic disease. In another embodiment, the compound is administeredafter initiation of ophthalmic disease.

Stroke

The compounds of the invention are used in methods of treating,preventing, ameliorating a stroke in a subject who is at risk ofsuffering a stroke, is suffering from a stroke or has suffered a stroke.The compounds of the invention are useful in methods of treatingpatients who are undergoing post-stroke rehabilitation.

A stroke, also known as a cerebrovascular accident (CVA), is an acuteneurological injury whereby the blood supply to a part of the brain isinterrupted due to either blockage of an artery or rupture of a bloodvessel. The part of the brain in which blood supply is interrupted nolonger receives oxygen and/or nutrients carried by the blood. The braincells become damaged or necrotic, thereby impairing function in or fromthat part of the brain. Brain tissue ceases to function if deprived ofoxygen for more than 60 to 90 seconds and after a few minutes willsuffer irreversible injury possibly leading to a death of the tissue,i.e., infarction.

Strokes are classified into two major types: ischemic, i.e., blockage ofa blood vessel supplying the brain, and hemorrhagic, i.e., bleeding intoor around the brain. The majority of all strokes are ischemic strokes.Ischemic stroke is commonly divided into thrombotic stroke, embolicstroke, systemic hypoperfusion (Watershed stroke), or venous thrombosis.In thrombotic stroke, a thrombus-forming process develops in theaffected artery, the thrombus, i.e., blood clot, gradually narrows thelumen of the artery, thereby impeding blood flow to distal tissue. Theseclots usually form around atherosclerotic plaques. There are two typesof thrombotic strokes, which are categorized based on the type of vesselon which the thrombus is formed. Large vessel thrombotic stroke involvesthe common and internal carotids, vertebral, and the Circle of Willis.Small vessel thrombotic stroke involves the intracerebral arteries,branches of the Circle of Willis, middle cerebral artery stem, andarteries arising from the distal vertebral and basilar artery.

A thrombus, even if non-occluding, can lead to an embolic stroke if thethrombus breaks off, at which point it becomes an embolus. An embolusrefers to a traveling particle or debris in the arterial bloodstreamoriginating from elsewhere. Embolic stroke refers to the blockage ofarterial access to a part of the brain by an embolus. An embolus isfrequently a blood clot, but it can also be a plaque that has broken offfrom an atherosclerotic blood vessel or a number of other substancesincluding fat, air, and even cancerous cells. Because an embolus arisesfrom elsewhere, local therapy only solves the problem temporarily. Thus,the source of the embolus must be identified. There are four categoriesof embolic stroke: those with a known cardiac source; those with apotential cardiac or aortic source (from trans-thoracic ortrans-esophageal echocardiogram); those with an arterial source; andthose with unknown source.

Systemic hypoperfusion is the reduction of blood flow to all parts ofthe body. It is most commonly due to cardiac pump failure from cardiacarrest or arrhythmias, or from reduced cardiac output as a result ofmyocardial infarction, pulmonary embolism, pericardial effusion, orbleeding. Hypoxemia (i.e., low blood oxygen content) may precipitate thehypoperfusion. Because the reduction in blood flow is global, all partsof the brain may be affected, especially the “watershed” areas which areborder zone regions supplied by the major cerebral arteries. Blood flowto these area has not necessary stopped, but instead may have lessenedto the point where brain damage occurs.

Veins in the brain function to drain the blood back to the body. Whenveins are occluded due to thrombosis, the draining of blood is blockedand the blood backs up, causing cerebral edema. This cerebral edema canresult in both ischemic and hemorrhagic strokes. This commonly occurs inthe rare disease sinus vein thrombosis.

Stroke is diagnosed in a subject or patient using one or more of avariety of techniques known in the art, such as, for example,neurological examination, blood tests, CT scans (without contrastenhancements), MRI scans, Doppler ultrasound, and arteriography (i.e.,roentgenography of arteries after injection of radiopacque material intothe blood stream). If a stroke is confirmed on imaging, various otherstudies are performed to determine whether there is a peripheral sourceof emboli. These studies include, e.g., an ultrasound/doppler study ofthe carotid arteries (to detect carotid stenosis); an electrocardiogram(ECG) and echocardiogram (to identify arrhythmias and resultant clots inthe heart which may spread to the brain vessels through thebloodstream); a Holter monitor study to identify intermittentarrhythmias and an angiogram of the cerebral vasculature (if a bleed isthought to have originated from an aneurysm or arteriovenousmalformation).

Compounds useful in these methods of treating, preventing orameliorating stroke or a symptom associated with stroke are compoundsthat modulate kinase signaling cascade proceeding, during or after astroke. In some embodiments, the compound is a kinase inhibitor. Forexample, the compound is a tyrosine kinase inhibitor. In an embodiment,the tyrosine kinase inhibitor is an Src inhibitor. For example, thecompound used in the methods of treating, preventing or amelioratingstroke or a symptom associated with stroke described herein is anallosteric inhibitor of kinase signaling cascade preceding, during orafter a stroke. Preferably, the compound used in the methods oftreating, preventing or ameliorating stroke or a symptom associated withstroke described herein is a non-ATP competitive inhibitor of kinasesignaling cascade preceding, during or after a stroke.

Inhibition of Src activity has been shown to provide cerebral protectionduring stroke. (See Paul et al., Nature Medicine, vol. 7(2):222-227(2001), which is hereby incorporated by reference in its entirety).Vascular endothelia growth factor (VEGF), which is produced in responseto the ischemic injury, has been shown to promote vascular permeability.Studies have shown that the Src kinase regulates VEGF-mediated VP in thebrain following stroke, and administration of an Src inhibitor beforeand after stroke reduced edema, improved cerebral perfusion anddecreased infarct volume after injury occurred. (Paul et al., 2001).Thus, Src inhibition may be useful in the prevention, treatment oramelioration of secondary damage following a stroke.

The compounds of the invention prevent, treat or ameliorate stroke or asymptom associated with stroke. Symptoms of a stroke include suddennumbness or weakness, especially on one side of the body; suddenconfusion or trouble speaking or understanding speech; sudden troubleseeing in one or both eyes; sudden trouble with walking, dizziness, orloss of balance or coordination; or sudden severe headache with no knowncause.

Generally there are three treatment stages for stroke: prevention,therapy immediately after the stroke, and post-stroke rehabilitation.Therapies to prevent a first or recurrent stroke are based on treatingthe underlying risk factors for stroke, such as, e.g., hypertension,high cholesterol, atrial fibrillation, and diabetes. Acute stroketherapies try to stop a stroke while it is happening by quicklydissolving the blood clot causing an ischemic stroke or by stopping thebleeding of a hemorrhagic stroke. Post-stroke rehabilitation helpsindividuals overcome disabilities that result from stroke damage.Medication or drug therapy is the most common treatment for stroke. Themost popular classes of drugs used to prevent or treat stroke areanti-thrombotics (e.g., anti-platelet agents and anticoagulants) andthrombolytics. The compounds are administered to a patient who is atrisk of suffering a stroke, is suffering from a stroke or has suffered astroke at a time before, during, after, or any combination thereof, theoccurrence of a stroke. The compounds of the invention are administeredalone, in pharmaceutical compositions, or in combination with any of avariety of known treatments, such as, for example, an anti-plateletmedication (e.g., aspirin, clopidogrel, dipyridamole), an anti-coagulant(e.g., warfarin), or a thrombolytic medication (e.g., tissue plasminogenactivator (t-PA), reteplase, Urokinase, streptokinase, tenectaplase,lanoteplase, or anistreplase.

Another aspect of the invention includes a method of protecting againstor treating stroke in a subject comprising administering a compositioncomprising an effective amount of a substantially pure KX2-391, or asalt, solvate, hydrate, or prodrug thereof, for example, substantiallypure KX2-391, KX2-391.2HCl, or KX2-391.MSA.

In one embodiment, the compound is administered before a stroke hasoccurred. In another embodiment, the compound is administered after astroke has occurred.

Atherosclerosis

The compounds of the invention are used in methods of treating,preventing, ameliorating atherosclerosis or a symptom thereof in asubject who is at risk for or suffering from atherosclerosis.

Atherosclerosis is a disease affecting the arterial blood vessel and iscommonly referred to as a “hardening” of the arteries. It is caused bythe formation of multiple plaques within the arteries. Atheroscleroticplaques, though compensated for by artery enlargement, eventually leadto plaque ruptures and stenosis (i.e., narrowing) of the artery, which,in turn, leads to an insufficient blood supply to the organ it feeds.Alternatively, if the compensating artery enlargement process isexcessive, a net aneurysm results. These complications are chronic,slowly progressing and cumulative. Most commonly, soft plaque suddenlyruptures, causing the formation of a blood clot (i.e., thrombus) thatrapidly slows or stops blood flow, which, in turn, leads to death of thetissues fed by the artery. This catastrophic event is called aninfarction. For example, coronary thrombosis of a coronary artery causesa myocardial infarction, commonly known as a heart attack. A myocardialinfarction occurs when an atherosclerotic plaque slowly builds up in theinner lining of a coronary artery and then suddenly ruptures, totallyoccluding the artery and preventing blood flow downstream.

Atherosclerosis and acute myocardial infarction are diagnosed in apatient using any of a variety of clinical and/or laboratory tests suchas, physical examination, radiologic or ultrasound examination and bloodanalysis. For example, a doctor or clinical can listen to a subject'sarteries to detect an abnormal whooshing sound, called a bruit. A bruitcan be heard with a stethoscope when placed over the affected artery.Alternatively, or in addition, the clinician or physician can checkpulses, e.g., in the leg or foot, for abnormalities such as weakness orabsence. The physician or clinical may perform blood work to check forcholesterol levels or to check the levels of cardiac enzymes, such ascreatine kinase, troponin and lactate dehydrogenase, to detectabnormalities. For example, troponin sub-units I or T, which are veryspecific for the myocardium, rise before permanent injury develops. Apositive troponin in the setting of chest pain may accurately predict ahigh likelihood of a myocardial infarction in the near future. Othertests to diagnose atherosclerosis and/or myocardial infarction include,for example, EKG (electrocardiogram) to measure the rate and regularityof a subject's heartbeat; chest X-ray, measuring ankle/brachial index,which compares the blood pressure in the ankle with the blood pressurein the arm; ultrasound analysis of arteries; CT scan of areas ofinterest; angiography; an exercise stress test, nuclear heart scanning;and magnetic resonance imaging (MRI) and positron emission tomography(PET) scanning of the heart.

Compounds useful in these methods of treating, preventing orameliorating atherosclerosis or a symptom thereof are compounds thatmodulate kinase signaling cascade in a patient at risk for or sufferingfrom atherosclerosis. In some embodiments, the compound is a kinaseinhibitor. For example, the compound is a tyrosine kinase inhibitor. Inan embodiment, the tyrosine kinase inhibitor is a Src inhibitor.Preferably, the compound used in the methods of treating, preventing orameliorating atherosclerosis or a symptom thereof described herein is anallosteric inhibitor of kinase signaling cascade involved inatherosclerosis. Preferably, the compound used in the methods oftreating, preventing or ameliorating atherosclerosis or a symptomassociated with atherosclerosis described herein is a non-ATPcompetitive inhibitor of kinase signaling cascade involved inatherosclerosis.

Cellular signal transduction by Src is believed to play a key role inincreased permeability of vessels, known as vascular permeability (VP).Vascular endothelia growth factor (VEGF), which is produced in responseto the ischemic injury, including, e.g., myocardial infarction, has beenshown to promote vascular permeability. Studies have shown that theinhibition of Src kinase decreases VEGF-mediated VP. (See Parang andSun, Expert Opin. Ther. Patents, vol. 15(9): 1183-1206 (2005), which ishereby incorporated by reference in its entirety). Mice treated with aSrc inhibitor demonstrated reduced tissue damage associated with traumaor injury to blood vessels after myocardial infarction, as compared tountreated mice. (See e.g., U.S. Patent Publication Nos. 20040214836 and20030130209 by Cheresh et al., the contents of which are herebyincorporated by reference in their entirety). Thus, Src inhibition maybe useful in the prevention, treatment or amelioration of secondarydamage following injury due to atherosclerosis, such as, for example,myocardial infarction.

Atherosclerosis generally does not produce symptoms until it severelynarrows the artery and restricts blood flow, or until it causes a suddenobstruction. Symptoms depend on where the plaques and narrowing develop,e.g., in the heart, brain, other vital organs and legs or almostanywhere in the body. The initial symptoms of atherosclerosis may bepain or cramps when the body requires more oxygen, for example duringexercise, when a person may feel chest pain (angina) because of lack ofoxygen to the heart or leg cramps because of lack of oxygen to the legs.Narrowing of the arteries supplying blood to the brain may causedizziness or transient ischemic attacks (TIA's) where the symptoms andsigns of a stroke last less than 24 hours. Typically, these symptomsdevelop gradually.

Symptoms of myocardial infarction are characterized by varying degreesof chest pain, discomfort, sweating, weakness, nausea, vomiting, andarrhythmias, sometimes causing loss of consciousness. Chest pain is themost common symptom of acute myocardial infarction and is oftendescribed as a tightness, pressure, or squeezing sensation. Pain mayradiate to the jaw, neck, arms, back, and epigastrium, most often to theleft arm or neck. Chest pain is more likely caused by myocardialinfarction when it lasts for more than 30 minutes. Patients sufferingfrom a myocardial infarction may exhibit shortness of breath (dyspnea)especially if the decrease in myocardial contractility due to theinfarct is sufficient to cause left ventricular failure with pulmonarycongestion or even pulmonary edema.

The compounds of the invention are administered alone, in pharmaceuticalcompositions, or in combination with any of a variety of knowntreatments for atherosclerosis, such as, for example,cholesterol-lowering drugs (e.g., statins), anti-platelet medications,or anti-coagulants.

Another aspect of the invention includes a method of protecting againstor treating athrosclerosis in a subject comprising administering acomposition comprising an effective amount of a substantially pureKX2-391, or a salt, solvate, hydrate, or prodrug thereof, for example,substantially pure KX2-391, KX2-391.2HCl, or KX2-391.MSA. In oneembodiment, the compound is administered before symptoms ofatherosclerosis occur. In another embodiment, the compound isadministered after the onset of symptoms of atherosclerosis.

Neuropathic Pain

The compounds of the invention are used in methods of treating,preventing, ameliorating neuropathic pain, such as chronic neuropathicpain, or a symptom thereof in a subject who is at risk of sufferingfrom, is suffering from, or has suffered neuropathic pain.

Neuropathic pain, also known as neuralgia, is qualitatively differentfrom ordinary nociceptive pain. Neuropathic pain usually presents as asteady burning and/or “pins and needles” and/or “electric shock”sensations. The difference between nociceptive pain and neuropathic painis due to the fact that “ordinary”, nociceptive pain stimulates onlypain nerves, while a neuropathy often results in the stimulation of bothpain and non-pain sensory nerves (e.g., nerves that respond to touch,warmth, cool) in the same area, thereby producing signals that thespinal cord and brain do not normally expect to receive.

Neuropathic pain is a complex, chronic pain state that usually isaccompanied by tissue injury. With neuropathic pain, the nerve fibersthemselves may be damaged, dysfunctional or injured. These damaged nervefibers send incorrect signals to other pain centers. The impact of nervefiber injury includes a change in nerve function both at the site ofinjury and areas around the injury.

Neuropathic pain is diagnosed in a subject or patient using one or moreof a variety of laboratory and/or clinical techniques known in the art,such as, for example, physical examination.

Compounds useful in these methods of treating, preventing orameliorating neuropathic pain, such as chronic neuropathic pain, or asymptom associated with neuropathic pain are compounds that modulatekinase signaling cascade involved in neuropathic pain.

c-Src has been shown to regulate the activity of N-methyl-D-aspartate(NMDA) receptors. (See Yu et al., Proc. Natl. Acad. Sci. USA, vol.96:7697-7704 (1999), which is hereby incorporated by reference in itsentirety). Studies have shown that PP2, a low molecular weight Srckinase inhibitor, decreases phosphorylation of the NMDA receptor NM2subunit. (See Guo et al., J. Neuro., vol. 22:6208-6217 (2002), which ishereby incorporated by reference in its entirety). Thus, Src inhibition,which in turn, inhibits the activity NMDA receptors, may be useful inthe prevention, treatment or amelioration of neuropathic pain, such aschronic neuropathic pain.

The compounds of the invention prevent, treat or ameliorate neuropathicpain, such as chronic neuropathic pain, or a symptom associated withneuropathic pain. Symptoms of neuropathic pain include shooting andburning pain, tingling and numbness.

The compounds of the invention are administered alone, in pharmaceuticalcompositions, or in combination with any of a variety of knowntreatments, such as, for example, analgesics, opioids, tricyclicantidepressants, anticonvulsants and serotonin norepinephrine reuptakeinhibitors.

In one embodiment, the compound is administered before the onset ofchronic neuropathic pain. In another embodiment, the compound isadministered after the onset of chronic neuropathic pain.

Hepatitis B

The compounds of the invention are used in methods of treating,preventing, ameliorating hepatitis B or a symptom thereof in a subjectwho is at risk for or suffering from hepatitis B.

The hepatitis B virus, a member of the Hepadnavirus family, consists ofa proteinaceous core particle containing the viral genome in the form ofdouble stranded DNA with single-stranded regions and an outerlipid-based envelope with embedded proteins. The envelope proteins areinvolved in viral binding and release into susceptible cells. The innercapsid relocates the DNA genome to the cell's nucleus where viral mRNAsare transcribed. Three subgenomic transcripts encoding the envelopeproteins are made, along with a transcript encoding the X protein. Afourth pre-genomic RNA is transcribed, which is exported to the cytosoland translates the viral polymerase and core proteins. Polymerase andpre-genomic RNA are encapsidated in assembling core particles, wherereverse transcription of the pre-genomic RNA to genomic DNA occurs bythe polymerase protein. The mature core particle then exits the cell vianormal secretory pathways, acquiring an envelope along the way.

Hepatitis B is one of a few known non-retroviral viruses that employreverse transcription as part of the replication process. Other viruseswhich use reverse transcription include, e.g., HTLV or HIV.

During HBV infection, the host immune response is responsible for bothhepatocellular damage and viral clearance. While the innate immuneresponse does not play a significant role in these processes, theadaptive immune response, particularly virus-specific cytotoxic Tlymphocytes (CTLs), contributes to nearly all of the liver injuryassociated with HBV infection. By killing infected cells and byproducing antiviral cytokines capable of purging HBV from viablehepatocytes, CTLs also eliminate the virus. Although liver damage isinitiated and mediated by the CTLs, antigen-nonspecific inflammatorycells can worsen CTL-induced immunopathology and platelets mayfacilitate the accumulation of CTLs into the liver.

Hepatitis B is diagnosed in a patient using any of a variety of clinicaland/or laboratory tests such as, physical examination, and blood orserum analysis. For example, blood or serum is assayed for the presenceof viral antigens and/or antibodies produced by the host. In a commontest for Hepatitis B, detection of hepatitis B surface antigen (HBsAg)is used to screen for the presence of infection. It is the firstdetectable viral antigen to appear during infection with this virus;however, early in an infection, this antigen may not be present and itmay be undetectable later in the infection as it is being cleared by thehost. During this ‘window’ in which the host remains infected but issuccessfully clearing the virus, IgM antibodies to the hepatitis B coreantigen (anti-HBc IGM) may be the only serologic evidence of disease.

Shortly after the appearance of the HBsAg, another antigen named as thehepatitis B e antigen (HBeAg) will appear. Traditionally, the presenceof HBeAg in a host's serum is associated with much higher rates of viralreplication; however, some variants of the hepatitis B virus do notproduce the “e” antigen at all. During the natural course of aninfection, the HBeAg may be cleared, and antibodies to the “e” antigen(anti-HBe) will arise immediately afterward. This conversion is usuallyassociated with a dramatic decline in viral replication. If the host isable to clear the infection, eventually the HBsAg will becomeundetectable and will be followed by antibodies to the hepatitis Bsurface antigen (anti-HBs). A person negative for HBsAg but positive foranti-HBs has either cleared an infection or has been vaccinatedpreviously. A number of people who are positive for HBsAg may have verylittle viral multiplication, and hence may be at little risk oflong-term complications or of transmitting infection to others.

Compounds useful in these methods of treating, preventing orameliorating hepatitis B or a symptom thereof are compounds thatmodulate kinase signaling cascade in a patient at risk for or sufferingfrom hepatitis B. In some embodiments, the compound is a kinaseinhibitor. For example, the compound is a tyrosine kinase inhibitor. Inan embodiment, the tyrosine kinase inhibitor is a Src inhibitor.Preferably, the compound used in the methods of treating, preventing orameliorating hepatitis B or a symptom thereof described herein is anallosteric inhibitor of kinase signaling cascade involved in hepatitisB. Preferably, the compound used in the methods of treating, preventingor ameliorating hepatitis B or a symptom associated with hepatitis Bdescribed herein is a non-ATP competitive inhibitor of kinase signalingcascade involved in hepatitis B.

Src plays a role in the replication of the hepatitis B virus. Thevirally encoded transcription factor HBx activates Src in a step that isrequired from propagation of the HBV virus. (See, e.g., Klein et al.,EMBO J., vol. 18:5019-5027 (1999); Klein et al., Mol. Cell. Biol., vol.17:6427-6436 (1997), each of which is hereby incorporated by referencein its entirety). Thus, Src inhibition, which in turn, inhibitsSrc-mediated propagation of the HBV virus, may be useful in theprevention, treatment or amelioration of hepatitis B or a symptomthereof

The compounds of the invention prevent, treat or ameliorate hepatitis Bor a symptom associated with hepatitis B. Symptoms of hepatitis Btypically develop within 30-180 days of exposure to the virus. However,up to half of all people infected with the hepatitis B virus have nosymptoms. The symptoms of hepatitis B are often compared to flu, andinclude, e.g., appetite loss; fatigue; nausea and vomiting, itching allover the body; pain over the liver (e.g., on the right side of theabdomen, under the lower rib cage), jaundice, and changes in excretoryfunctions.

The compounds of the invention are administered alone, in pharmaceuticalcompositions, or in combination with any of a variety of knowntreatments for hepatitis B, such as, for example, interferon alpha,lamivudine (Epivir-HBV) and baraclude (entecavir).

Another aspect of the invention includes a method of protecting againstor treating hepatitis B in a subject comprising administering acomposition comprising an effective amount of a substantially pureKX2-391, or a salt, solvate, hydrate, or prodrug thereof, for example,substantially pure KX2-391, KX2-391.2HCl, or KX2-391.MSA.

In one embodiment, the compound is administered before the subject hascontracted hepatitis B. In another embodiment, the compound isadministered after the subject has contracted hepatitis B.

Regulation of Immune System Activity

As described herein, the compounds of the invention may be used toregulate immune system activity in a subject, thereby protecting againstor preventing autoimmune disease, e.g., rheumatoid arthritis, multiplesclerosis, sepsis and lupus as well as transplant rejection and allergicdiseases. Alternatively, the compound may be used to treat autoimmunedisease in a subject. For example, the compound may result in reductionin the severity of symptoms or halt impending progression of theautoimmune disease in a subject. The compound of the invention may beinvolved in modulating a kinase signaling cascade, e.g., a kinaseinhibitor, a non-ATP competitive inhibitor, a tyrosine kinase inhibitor,e.g., a Src inhibitor, a p59fyn (Fyn) inhibitor or a p56lck (Lck)inhibitor.

Autoimmune diseases are diseases caused by a breakdown of self-tolerancesuch that the adaptive immune system responds to self antigens andmediates cell and tissue damage. Autoimmune diseases can be organspecific (e.g., thyroiditis or diabetes) or systemic (e.g., systemiclupus erythematosus). T cells modulate the cell-mediated immune responsein the adaptive immune system. Under normal conditions, T cells expressantigen receptors (T cell receptors) that recognize peptide fragments offoreign proteins bound to self major histocompatibility complexmolecules. Among the earliest recognizable events after T cell receptor(TCR) stimulation are the activation of Lck and Fyn, resulting in TCRphosphorylation on tyrosine residues within immunoreceptortyrosine-based activation motifs (Zamoyska, et al.; 2003, Immunol. Rev.,191, 107-118). Tyrosine kinases, such as Lck (which is a member of theSrc family of protein tyrosine kinases) play an essential role in theregulation of cell signaling and cell proliferation by phosphorylatingtyrosine residues of peptides and proteins (Levitzki; 2001, Top. Curr.Chem., 211, 1-15; Longati, et al.; 2001, Curr. Drug Targets, 2, 41-55;Qian, and Weiss; 1997, Curr. Opin. Cell Biol., 9, 205-211). Thus,although not wishing to be bound by theory, it is hypothesized that theadministration of a compound of the instant invention which modulatestyrosine kinase (e.g., Src) activity is useful in the treatment ofautoimmune disease.

The tyrosine kinases lck and fyn are both activated in the TCR pathway;thus, inhibitors of lck and/or fyn have potential utility as autoimmuneagents (Palacios and Weiss; 2004, Oncogene, 23, 7990-8000). Lck and Fynare predominantly expressed by T cells through most of their lifespan.The roles of Lck and Fyn in T cell development, homeostasis andactivation have been demonstrated by animal and cell line studies(Parang and Sun; 2005, Expert Opin. The. Patents, 15, 1183-1207). Lckactivation is involved in autoimmune diseases and transplant rejection(Kamens, et al.; 2001, Curr. Opin. Investig. Drugs, 2, 1213-1219).Results have shown that the lck (−) Jurkat cell lines are unable toproliferate, produce cytokines, and generate increases in intracellularcalcium, inositol phosphate, and tyrosine phosphorylation in response toT cell receptor stimulation (Straus and Weiss; 1992, Cell., 70, 585-593;Yamasaki, et al.; 1996, Mol. Cell. Biol., 16, 7151-7160). Therefore, anagent inhibiting lck would effectively block T cell function, act as animmunosuppressive agent, and have potential utility in autoimmunediseases, such as rheumatoid arthritis, multiple sclerosis, and lupus,as well as in the area of transplant rejection and allergic diseases(Hanke and Pollok; 1995, Inflammation Res., 44, 357-371). Thus, althoughnot wishing to be bound by theory, it is hypothesized that theadministration of a compound of the instant invention which modulatesone or more members of the Src family of protein tyrosine kinases (e.g.,lck and/or fyn) is useful in the treatment of autoimmune disease.

Another aspect of the invention includes a method of regulating immunesystem activity in a subject comprising administering a compositioncomprising an effective amount of a substantially pure KX2-391, or asalt, solvate, hydrate, or prodrug thereof, for example, substantiallypure KX2-391, KX2-391.2HCl, or KX2-391.MSA.

DEFINITIONS

For convenience, certain terms used in the specification, examples andappended claims are collected here.

Protein kinases are a large class of enzymes which catalyze the transferof the γ-phosphate from ATP to the hydroxyl group on the side chain ofSer/Thr or Tyr in proteins and peptides and are intimately involved inthe control of various important cell functions, perhaps most notably:signal transduction, differentiation, and proliferation. There areestimated to be about 2,000 distinct protein kinases in the human body,and although each of these phosphorylates particular protein/peptidesubstrates, they all bind the same second substrate ATP in a highlyconserved pocket. About 50% of the known oncogene products are proteintyrosine kinases (PTKs), and their kinase activity has been shown tolead to cell transformation.

The PTKs can be classified into two categories, the membrane receptorPTKs (e.g. growth factor receptor PTKs) and the non-receptor PTKs (e.g.the Src family of proto-oncogene products and focal adhesion kinase(FAK)). The hyperactivation of Src has been reported in a number ofhuman cancers, including those of the colon, breast, lung, bladder, andskin, as well as in gastric cancer, hairy cell leukemia, andneuroblastoma.

“Inhibits one or more components of a protein kinase signaling cascade”means that one or more components of the kinase signaling cascade areeffected such that the functioning of the cell changes. Components of aprotein kinase signaling cascade include any proteins involved directlyor indirectly in the kinase signaling pathway including secondmessengers and upstream and downstream targets.

“Treating”, includes any effect, e.g., lessening, reducing, modulating,or eliminating, that results in the improvement of the condition,disease, disorder, etc. “Treating” or “treatment” of a disease stateincludes: inhibiting the disease state, i.e., arresting the developmentof the disease state or its clinical symptoms; or relieving the diseasestate, i.e., causing temporary or permanent regression of the diseasestate or its clinical symptoms.

“Preventing” the disease state includes causing the clinical symptoms ofthe disease state not to develop in a subject that may be exposed to orpredisposed to the disease state, but does not yet experience or displaysymptoms of the disease state.

“Disease state” means any disease, disorder, condition, symptom, orindication.

As used herein, the term “cell proliferative disorder” refers toconditions in which the unregulated and/or abnormal growth of cells canlead to the development of an unwanted condition or disease, which canbe cancerous or non-cancerous, for example a psoriatic condition. Asused herein, the terms “psoriatic condition” or “psoriasis” refers todisorders involving keratinocyte hyperproliferation, inflammatory cellinfiltration, and cytokine alteration.

In one embodiment, the cell proliferation disorder is cancer. As usedherein, the term “cancer” includes solid tumors, such as lung, breast,colon, ovarian, brain, liver, pancreas, prostate, malignant melanoma,non-melanoma skin cancers, as well as hematologic tumors and/ormalignancies, such as childhood leukemia and lymphomas, multiplemyeloma, Hodgkin's disease, lymphomas of lymphocytic and cutaneousorigin, acute and chronic leukemia such as acute lymphoblastic, acutemyelocytic or chronic myelocytic leukemia, plasma cell neoplasm,lymphoid neoplasm and cancers associated with AIDS.

In addition to psoriatic conditions, the types of proliferative diseaseswhich may be treated using the compositions of the present invention areepidermic and dermoid cysts, lipomas, adenomas, capillary and cutaneoushemangiomas, lymphangiomas, nevi lesions, teratomas, nephromas,myofibromatosis, osteoplastic tumors, and other dysplastic masses andthe like. The proliferative diseases can include dysplasias anddisorders of the like.

“A therapeutically effective amount” means the amount of a compoundthat, when administered to a mammal for treating a disease, issufficient to effect such treatment for the disease. In one embodiment,a therapeutically effective amount is administered to a mammal to reducethe level of the disease e.g., to reduce the level of hearing loss. Inone embodiment, a therapeutically effective amount of a compound isadministered. In another embodiment, therapeutically effective amount ofa composition is administered. The “therapeutically effective amount”will vary depending on the compound, the disease and its severity andthe age, weight, etc., of the mammal to be treated.

A therapeutically effective amount of one or more of the compounds canbe formulated with a pharmaceutically acceptable carrier foradministration to a human or an animal. Accordingly, the compounds orthe formulations can be administered, for example, via oral, parenteral,or topical routes, to provide a therapeutically effective amount of thecompound. In alternative embodiments, the compounds prepared inaccordance with the present invention can be used to coat or impregnatea medical device, e.g., a stent.

The term “prophylactically effective amount” means an effective amountof a compound or compounds, of the present invention that isadministered to effect prevention of the disease. In one embodiment, aprophylactically effective amount of a compound is administered. Inanother embodiment, prophylactically effective amount of a compositionis administered.

“Pharmacological effect” as used herein encompasses effects produced inthe subject that achieve the intended purpose of a therapy. In oneembodiment, a pharmacological effect means that primary indications ofthe subject being treated are prevented, alleviated, or reduced. Forexample, a pharmacological effect would be one that results in theprevention, alleviation or reduction of primary indications in a treatedsubject. In another embodiment, a pharmacological effect means thatdisorders or symptoms of the primary indications of the subject beingtreated are prevented, alleviated, or reduced. For example, apharmacological effect would be one that results in the prevention orreduction of primary indications in a treated subject.

Compounds of the present invention that contain nitrogens can beconverted to N-oxides by treatment with an oxidizing agent (e.g.,3-chloroperoxybenzoic acid (m-CPBA) and/or hydrogen peroxides) to affordother compounds of the present invention. Thus, all shown and claimednitrogen-containing compounds are considered, when allowed by valencyand structure, to include both the compound as shown and its N-oxidederivative (which can be designated as N→O or N⁺—O⁻). Furthermore, inother instances, the nitrogens in the compounds of the present inventioncan be converted to N-hydroxy or N-alkoxy compounds. For example,N-hydroxy compounds can be prepared by oxidation of the parent amine byan oxidizing agent such as m-CPBA. All shown and claimednitrogen-containing compounds are also considered, when allowed byvalency and structure, to cover both the compound as shown and itsN-hydroxy (i.e., N—OH) and N-alkoxy (i.e., N—OR, wherein R issubstituted or unsubstituted C₁₋₆ alkyl, C₁₋₆ alkenyl, C₁₋₆ alkynyl,C₃₋₁₄ carbocycle, or 3-14-membered heterocycle) derivatives.

“Counterion” is used to represent a small, negatively charged speciessuch as chloride, bromide, hydroxide, acetate, and sulfate.

An “anionic group,” as used herein, refers to a group that is negativelycharged at physiological pH. Anionic groups include carboxylate,sulfate, sulfonate, sulfonate, sulfamate, tetrazolyl, phosphate,phosphonate, phosphinate, or phosphorothioate or functional equivalentsthereof. “Functional equivalents” of anionic groups are intended toinclude bioisosteres, e.g., bioisosteres of a carboxylate group.Bioisosteres encompass both classical bioisosteric equivalents andnon-classical bioisosteric equivalents. Classical and non-classicalbioisosteres are known in the art (see, e.g., Silverman, R. B. TheOrganic Chemistry of Drug Design and Drug Action, Academic Press, Inc.:San Diego, Calif., 1992, pp. 19-23). In one embodiment, an anionic groupis a carboxylate.

The present invention is intended to include all isotopes of atomsoccurring in the present compounds. Isotopes include those atoms havingthe same atomic number but different mass numbers. By way of generalexample and without limitation, isotopes of hydrogen include tritium anddeuterium, and isotopes of carbon include C-13 and C-14.

The compounds described herein may have asymmetric centers. Compounds ofthe present invention containing an asymmetrically substituted atom maybe isolated in optically active or racemic forms. It is well known inthe art how to prepare optically active forms, such as by resolution ofracemic forms or by synthesis from optically active starting materials.Many geometric isomers of olefins, C═N double bonds, and the like canalso be present in the compounds described herein, and all such stableisomers are contemplated in the present invention. Cis and transgeometric isomers of the compounds of the present invention aredescribed and may be isolated as a mixture of isomers or as separatedisomeric forms. All chiral, diastereomeric, racemic, and geometricisomeric forms of a structure are intended, unless the specificstereochemistry or isomeric form is specifically indicated. Alltautomers of shown or described compounds are also considered to be partof the present invention.

In the present specification, the structural formula of the compoundrepresents a certain isomer for convenience in some cases, but thepresent invention includes all isomers such as geometrical isomer,optical isomer based on an asymmetrical carbon, stereoisomer, tautomerand the like which occur structurally and an isomer mixture and is notlimited to the description of the formula for convenience, and may beany one of isomer or a mixture. Therefore, an asymmetrical carbon atommay be present in the molecule and an optically active compound and aracemic compound may be present in the present compound, but the presentinvention is not limited to them and includes any one. In addition, acrystal polymorphism may be present but is not limiting, but any crystalform may be single or a crystal form mixture, or an anhydride orhydrate. Further, so-called metabolite which is produced by degradationof the present compound in vivo is included in the scope of the presentinvention.

“Isomerism” means compounds that have identical molecular formulae butthat differ in the nature or the sequence of bonding of their atoms orin the arrangement of their atoms in space. Isomers that differ in thearrangement of their atoms in space are termed “stereoisomers”.Stereoisomers that are not mirror images of one another are termed“diastereoisomers”, and stereoisomers that are non-superimposable mirrorimages are termed “enantiomers”, or sometimes optical isomers. A carbonatom bonded to four nonidentical substituents is termed a “chiralcenter”.

“Chiral isomer” means a compound with at least one chiral center. It hastwo enantiomeric forms of opposite chirality and may exist either as anindividual enantiomer or as a mixture of enantiomers. A mixturecontaining equal amounts of individual enantiomeric forms of oppositechirality is termed a “racemic mixture”. A compound that has more thanone chiral center has 2^(n-1)enantiomeric pairs, where n is the numberof chiral centers. Compounds with more than one chiral center may existas either an individual diastereomer or as a mixture of diastereomers,termed a “diastereomeric mixture”. When one chiral center is present, astereoisomer may be characterized by the absolute configuration (R or S)of that chiral center. Absolute configuration refers to the arrangementin space of the substituents attached to the chiral center. Thesubstituents attached to the chiral center under consideration areranked in accordance with the Sequence Rule of Cahn, Ingold and Prelog.(Calm et al, Angew. Chem. Inter. Edit. 1966, 5, 385; errata 511; Cahn etal., Angew. Chem. 1966, 78, 413; Calm and Ingold, J. Chem. Soc. 1951(London), 612; Cahn et al., Experientia 1956, 12, 81; Cahn, J., Chem.Educ. 1964, 41, 116).

“Geometric Isomers” means the diastereomers that owe their existence tohindered rotation about double bonds. These configurations aredifferentiated in their names by the prefixes cis and trans, or Z and E,which indicate that the groups are on the same or opposite side of thedouble bond in the molecule according to the Cahn-Ingold-Prelog rules.

Further, the structures and other compounds discussed in thisapplication include all atropic isomers thereof. “Atropic isomers” are atype of stereoisomer in which the atoms of two isomers are arrangeddifferently in space. Atropic isomers owe their existence to arestricted rotation caused by hindrance of rotation of large groupsabout a central bond. Such atropic isomers typically exist as a mixture,however as a result of recent advances in chromatography techniques, ithas been possible to separate mixtures of two atropic isomers in selectcases.

The terms “crystal polymorphs” or “polymorphs” or “crystal forms” meanscrystal structures in which a compound (or salt or solvate thereof) cancrystallize in different crystal packing arrangements, all of which havethe same elemental composition. Different crystal forms usually havedifferent X-ray diffraction patterns, infrared spectral, melting points,density hardness, crystal shape, optical and electrical properties,stability and solubility. Recrystallization solvent, rate ofcrystallization, storage temperature, and other factors may cause onecrystal form to dominate. Crystal polymorphs of the compounds can beprepared by crystallization under different conditions.

Additionally, the compounds of the present invention, for example, thesalts of the compounds, can exist in either hydrated or unhydrated (theanhydrous) form or as solvates with other solvent molecules. Nonlimitingexamples of hydrates include monohydrates, dihydrates, etc. Nonlimitingexamples of solvates include ethanol solvates, acetone solvates, etc.

“Solvates” means solvent addition forms that contain eitherstoichiometric or non stoichiometric amounts of solvent. Some compoundshave a tendency to trap a fixed molar ratio of solvent molecules in thecrystalline solid state, thus forming a solvate. If the solvent is waterthe solvate formed is a hydrate, when the solvent is alcohol, thesolvate formed is an alcoholate. Hydrates are formed by the combinationof one or more molecules of water with one of the substances in whichthe water retains its molecular state as H₂O, such combination beingable to form one or more hydrate.

“Tautomers” refers to compounds whose structures differ markedly inarrangement of atoms, but which exist in easy and rapid equilibrium. Itis to be understood that the compounds of the invention may be depictedas different tautomers. It should also be understood that when compoundshave tautomeric forms, all tautomeric forms are intended to be withinthe scope of the invention, and the naming of the compounds does notexclude any tautomer form.

Some compounds of the present invention can exist in a tautomeric form.Tautomers are also intended to be encompassed within the scope of thepresent invention.

The compounds, salts and prodrugs of the present invention can exist inseveral tautomeric forms, including the enol and imine form, and theketo and enamine form and geometric isomers and mixtures thereof. Allsuch tautomeric forms are included within the scope of the presentinvention. Tautomers exist as mixtures of a tautomeric set in solution.In solid form, usually one tautomer predominates. Even though onetautomer may be described, the present invention includes all tautomersof the present compounds

A tautomer is one of two or more structural isomers that exist inequilibrium and are readily converted from one isomeric form to another.This reaction results in the formal migration of a hydrogen atomaccompanied by a switch of adjacent conjugated double bonds. Insolutions where tautomerization is possible, a chemical equilibrium ofthe tautomers will be reached. The exact ratio of the tautomers dependson several factors, including temperature, solvent, and pH. The conceptof tautomers that are interconvertable by tautomerizations is calledtautomerism.

Of the various types of tautomerism that are possible, two are commonlyobserved. In keto-enol tautomerism a simultaneous shift of electrons anda hydrogen atom occurs. Ring-chain tautomerism, is exhibited by glucose.It arises as a result of the aldehyde group (—CHO) in a sugar chainmolecule reacting with one of the hydroxy groups (—OH) in the samemolecule to give it a cyclic (ring-shaped) form.

Tautomerizations are catalyzed by: Base: 1. deprotonation; 2. formationof a delocalized anion (e.g. an enolate); 3. protonation at a differentposition of the anion; Acid: 1. protonation; 2. formation of adelocalized cation; 3. deprotonation at a different position adjacent tothe cation.

Common tautomeric pairs are: ketone-enol, amide-nitrile, lactam-lactim,amide-imidic acid tautomerism in heterocyclic rings (e.g. in thenucleobases guanine, thymine, and cytosine), amine-enamine andenamine-enamine.

It is to be understood accordingly that the isomers arising fromasymmetric carbon atoms (e.g., all enantiomers and diastereomers) areincluded within the scope of the invention, unless indicated otherwise.Such isomers can be obtained in substantially pure form by classicalseparation techniques and by stereochemically controlled synthesis.Furthermore, the structures and other compounds and moieties discussedin this application also include all tautomers thereof. Alkenes caninclude either the E- or Z-geometry, where appropriate. The compounds ofthis invention may exist in stereoisomeric form, therefore can beproduced as individual stereoisomers or as mixtures.

A “pharmaceutical composition” is a formulation containing the disclosedcompounds in a form suitable for administration to a subject. In oneembodiment, the pharmaceutical composition is in bulk or in unit dosageform. It is can be advantageous to formulate compositions in dosage unitform for ease of administration and uniformity of dosage. Dosage unitform as used herein refers to physically discrete units suited asunitary dosages for the subject to be treated; each unit containing apredetermined quantity of active reagent calculated to produce thedesired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on the uniquecharacteristics of the active reagent and the particular therapeuticeffect to be achieved, and the limitations inherent in the art ofcompounding such an active agent for the treatment of individuals.

The unit dosage form is any of a variety of forms, including, forexample, a capsule, an IV bag, a tablet, a single pump on an aerosolinhaler, or a vial. The quantity of active ingredient (e.g., aformulation of the disclosed compound or salt, hydrate, solvate, orisomer thereof) in a unit dose of composition is an effective amount andis varied according to the particular treatment involved. One skilled inthe art will appreciate that it is sometimes necessary to make routinevariations to the dosage depending on the age and condition of thepatient. The dosage will also depend on the route of administration. Avariety of routes are contemplated, including oral, pulmonary, rectal,parenteral, transdermal, subcutaneous, intravenous, intramuscular,intraperitoneal, inhalational, buccal, sublingual, intrapleural,intrathecal, intranasal, and the like. Dosage forms for the topical ortransdermal administration of a compound of this invention includepowders, sprays, ointments, pastes, creams, lotions, gels, solutions,patches and inhalants. In one embodiment, the active compound is mixedunder sterile conditions with a pharmaceutically acceptable carrier, andwith any preservatives, buffers, or propellants that are required.

The term “flash dose” refers to compound formulations that are rapidlydispersing dosage forms.

The term “immediate release” is defined as a release of compound from adosage form in a relatively brief period of time, generally up to about60 minutes. The term “modified release” is defined to include delayedrelease, extended release, and pulsed release. The term “pulsed release”is defined as a series of releases of drug from a dosage form. The term“sustained release” or “extended release” is defined as continuousrelease of a compound from a dosage form over a prolonged period.

A “subject” includes mammals, e.g., humans, companion animals (e.g.,dogs, cats, birds, and the like), farm animals (e.g., cows, sheep, pigs,horses, fowl, and the like) and laboratory animals (e.g., rats, mice,guinea pigs, birds, and the like). In one embodiment, the subject ishuman.

As used herein, the phrase “pharmaceutically acceptable” refers to thosecompounds, materials, compositions, carriers, and/or dosage forms whichare, within the scope of sound medical judgment, suitable for use incontact with the tissues of human beings and animals without excessivetoxicity, irritation, allergic response, or other problem orcomplication, commensurate with a reasonable benefit/risk ratio.

“Pharmaceutically acceptable excipient” means an excipient that isuseful in preparing a pharmaceutical composition that is generally safe,non-toxic and neither biologically nor otherwise undesirable, andincludes excipient that is acceptable for veterinary use as well ashuman pharmaceutical use. A “pharmaceutically acceptable excipient” asused in the specification and claims includes both one and more than onesuch excipient.

The compounds of the invention are capable of further forming salts. Allof these forms are also contemplated within the scope of the claimedinvention.

“Pharmaceutically acceptable salt” of a compound means a salt that ispharmaceutically acceptable and that possesses the desiredpharmacological activity of the parent compound.

As used herein, “pharmaceutically acceptable salts” refer to derivativesof the disclosed compounds wherein the parent compound is modified bymaking acid or base salts thereof. Examples of pharmaceuticallyacceptable salts include, but are not limited to, mineral or organicacid salts of basic residues such as amines, alkali or organic salts ofacidic residues such as carboxylic acids, and the like. Thepharmaceutically acceptable salts include the conventional non-toxicsalts or the quaternary ammonium salts of the parent compound formed,for example, from non-toxic inorganic or organic acids. For example,such conventional non-toxic salts include, but are not limited to, thosederived from inorganic and organic acids selected from 2-acetoxybenzoic,2-hydroxyethane sulfonic, acetic, ascorbic, benzene sulfonic, benzoic,bicarbonic, carbonic, citric, edetic, ethane disulfonic, 1,2-ethanesulfonic, fumaric, glucoheptonic, gluconic, glutamic, glycolic,glycollyarsanilic, hexylresorcinic, hydrabamic, hydrobromic,hydrochloric, hydroiodic, hydroxymaleic, hydroxynaphthoic, isethionic,lactic, lactobionic, lauryl sulfonic, maleic, malic, mandelic, methanesulfonic, napsylic, nitric, oxalic, pamoic, pantothenic, phenylacetic,phosphoric, polygalacturonic, propionic, salicyclic, stearic, subacetic,succinic, sulfamic, sulfanilic, sulfuric, tannic, tartaric, toluenesulfonic, and the commonly occurring amine acids, e.g., glycine,alanine, phenylalanine, arginine, etc.

Other examples include hexanoic acid, cyclopentane propionic acid,pyruvic acid, malonic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamicacid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid,4-toluenesulfonic acid, camphorsulfonic acid,4-methylbicyclo-[2.2.2]-oct-2-ene-1-carboxylic acid, 3-phenylpropionicacid, trimethylacetic acid, tertiary butylacetic acid, muconic acid, andthe like. The invention also encompasses salts formed when an acidicproton present in the parent compound either is replaced by a metal ion,e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion; orcoordinates with an organic base such as ethanolamine, diethanolamine,triethanolamine, tromethamine, N-methylglucamine, and the like.

It should be understood that all references to pharmaceuticallyacceptable salts include solvent addition forms (solvates) or crystalforms (polymorphs) as defined herein, of the same salt.

The pharmaceutically acceptable salts of the present invention can besynthesized from a parent compound that contains a basic or acidicmoiety by conventional chemical methods. Generally, such salts can beprepared by reacting the free acid or base forms of these compounds witha stoichiometric amount of the appropriate base or acid in water or inan organic solvent, or in a mixture of the two; non-aqueous media likeether, ethyl acetate, ethanol, isopropanol, or acetonitrile can be used.Lists of suitable salts are found in Remington's PharmaceuticalSciences, 18th ed. (Mack Publishing Company, 1990). For example, saltscan include, but are not limited to, the hydrochloride and acetate saltsof the aliphatic amine-containing, hydroxylamine-containing, andimine-containing compounds of the present invention.

The compounds of the present invention can be prepared as prodrugs, forexample pharmaceutically acceptable prodrugs. The terms “pro-drug” and“prodrug” are used interchangeably herein and refer to any compoundwhich releases an active parent drug in vivo. Since prodrugs are knownto enhance numerous desirable qualities of pharmaceuticals (e.g.,solubility, bioavailability, manufacturing, etc.) the compounds of thepresent invention can be delivered in prodrug form. Thus, the presentinvention is intended to cover prodrugs of the presently claimedcompounds, methods of delivering the same and compositions containingthe same. “Prodrugs” are intended to include any covalently bondedcarriers that release an active parent drug of the present invention invivo when such prodrug is administered to a subject. Prodrugs thepresent invention are prepared by modifying functional groups present inthe compound in such a way that the modifications are cleaved, either inroutine manipulation or in vivo, to the parent compound. Prodrugsinclude compounds of the present invention wherein a hydroxy, amino,sulfhydryl, carboxy, or carbonyl group is bonded to any group that, maybe cleaved in vivo to form a free hydroxyl, free amino, free sulfhydryl,free carboxy or free carbonyl group, respectively.

Examples of prodrugs include, but are not limited to, esters (e.g.,acetate, dialkylaminoacetates, formates, phosphates, sulfates, andbenzoate derivatives) and carbamates (e.g., N,N-dimethylaminocarbonyl)of hydroxy functional groups, esters groups (e.g. ethyl esters,morpholinoethanol esters) of carboxyl functional groups, N-acylderivatives (e.g. N-acetyl) N-Mannich bases, Schiff bases and enaminonesof amino functional groups, oximes, acetals, ketals and enol esters ofketone and aldehyde functional groups in compounds, and the like, SeeBundegaard, H. “Design of Prodrugs” p1-92, Elesevier, New York-Oxford(1985).

“Protecting group” refers to a grouping of atoms that when attached to areactive group in a molecule masks, reduces or prevents that reactivity.Examples of protecting groups can be found in Green and Wuts, ProtectiveGroups in Organic Chemistry, (Wiley, 2^(nd) ed. 1991); Harrison andHarrison et al., Compendium of Synthetic Organic Methods, Vols. 1-8(John Wiley and Sons, 1971-1996); and Kocienski, Protecting Groups,(Verlag, 3^(rd) ed. 2003).

“Stable compound” and “stable structure” are meant to indicate acompound that is sufficiently robust to survive isolation to a usefuldegree of purity from a reaction mixture, and formulation into anefficacious therapeutic agent.

In the specification, the singular forms also include the plural, unlessthe context clearly dictates otherwise. Unless defined otherwise, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs. In the case of conflict, the present specificationwill control.

All percentages and ratios used herein, unless otherwise indicated, areby weight.

“Combination therapy” (or “co-therapy”) includes the administration of acompound of the invention and at least a second agent as part of aspecific treatment regimen intended to provide the beneficial effectfrom the co-action of these therapeutic agents. The beneficial effect ofthe combination includes, but is not limited to, pharmacokinetic orpharmacodynamic co-action resulting from the combination of therapeuticagents. Administration of these therapeutic agents in combinationtypically is carried out over a defined time period (usually minutes,hours, days or weeks depending upon the combination selected).“Combination therapy” may, but generally is not, intended to encompassthe administration of two or more of these therapeutic agents as part ofseparate monotherapy regimens that incidentally and arbitrarily resultin the combinations of the present invention.

“Combination therapy” is intended to embrace administration of thesetherapeutic agents in a sequential manner, that is, wherein eachtherapeutic agent is administered at a different time, as well asadministration of these therapeutic agents, or at least two of thetherapeutic agents, in a substantially simultaneous manner.Substantially simultaneous administration can be accomplished, forexample, by administering to the subject a single capsule having a fixedratio of each therapeutic agent or in multiple, single capsules for eachof the therapeutic agents. Sequential or substantially simultaneousadministration of each therapeutic agent can be effected by anyappropriate route including, but not limited to, oral routes,intravenous routes, intramuscular routes, and direct absorption throughmucous membrane tissues. The therapeutic agents can be administered bythe same route or by different routes. For example, a first therapeuticagent of the combination selected may be administered by intravenousinjection while the other therapeutic agents of the combination may beadministered orally. Alternatively, for example, all therapeutic agentsmay be administered orally or all therapeutic agents may be administeredby intravenous injection. The sequence in which the therapeutic agentsare administered is not narrowly critical.

“Combination therapy” also embraces the administration of thetherapeutic agents as described above in further combination with otherbiologically active ingredients and non-drug therapies (e.g., surgery orradiation treatment). Where the combination therapy further comprises anon-drug treatment, the non-drug treatment may be conducted at anysuitable time so long as a beneficial effect from the co-action of thecombination of the therapeutic agents and non-drug treatment isachieved. For example, in appropriate cases, the beneficial effect isstill achieved when the non-drug treatment is temporally removed fromthe administration of the therapeutic agents, perhaps by days or evenweeks.

Throughout the description, where compositions are described as having,including, or comprising specific components, it is contemplated thatcompositions also consist essentially of, or consist of the recitedcomponents. Similarly, where processes are described as having,including, or comprising specific process steps, the processes alsoconsist essentially of, or consist of, the recited processing steps.Further, it should be understood that the order of steps or order forperforming certain actions are immaterial so long as the inventionremains operable. Moreover, two or more steps or actions may beconducted simultaneously.

The compounds, or pharmaceutically acceptable salts thereof, isadministered orally, nasally, transdermally, pulmonary, inhalationally,buccally, sublingually, intraperintoneally, subcutaneously,intramuscularly, intravenously, rectally, intrapleurally, intrathecallyand parenterally. In one embodiment, the compound is administeredorally. One skilled in the art will recognize the advantages of certainroutes of administration.

The dosage regimen utilizing the compounds is selected in accordancewith a variety of factors including type, species, age, weight, sex andmedical condition of the patient; the severity of the condition to betreated; the route of administration; the renal and hepatic function ofthe patient; and the particular compound or salt thereof employed. Anordinarily skilled physician or veterinarian can readily determine andprescribe the effective amount of the drug required to prevent, counteror arrest the progress of the condition.

Techniques for formulation and administration of the disclosed compoundsof the invention can be found in Remington: the Science and Practice ofPharmacy, 19^(th) edition, Mack Publishing Co., Easton, Pa. (1995). Inan embodiment, the compounds described herein, and the pharmaceuticallyacceptable salts thereof, are used in pharmaceutical preparations incombination with a pharmaceutically acceptable carrier or diluent.Suitable pharmaceutically acceptable carriers include inert solidfillers or diluents and sterile aqueous or organic solutions. Thecompounds will be present in such pharmaceutical compositions in amountssufficient to provide the desired dosage amount in the range describedherein.

In one embodiment, the compound is prepared for oral administration,wherein the disclosed compounds or salts thereof are combined with asuitable solid or liquid carrier or diluent to form capsules, tablets,pills, powders, syrups, solutions, suspensions and the like.

The tablets, pills, capsules, and the like contain from about 1 to about99 weight percent of the active ingredient and a binder such as gumtragacanth, acacias, corn starch or gelatin; excipients such asdicalcium phosphate; a disintegrating agent such as corn starch, potatostarch or alginic acid; a lubricant such as magnesium stearate; and/or asweetening agent such as sucrose, lactose, saccharin, xylitol, and thelike. When a dosage unit form is a capsule, it often contains, inaddition to materials of the above type, a liquid carrier such as afatty oil.

In some embodiments, various other materials are present as coatings orto modify the physical form of the dosage unit. For instance, in someembodiments, tablets are coated with shellac, sugar or both. In someembodiments, a syrup or elixir contains, in addition to the activeingredient, sucrose as a sweetening agent, methyl and propylparabens aspreservatives, a dye and a flavoring such as cherry or orange flavor,and the like.

For some embodiments relating to parental administration, the disclosedcompounds, or salts, solvates, tautomers or polymorphs thereof, can becombined with sterile aqueous or organic media to form injectablesolutions or suspensions. In one embodiment, injectable compositions areaqueous isotonic solutions or suspensions. The compositions may besterilized and/or contain adjuvants, such as preserving, stabilizing,wetting or emulsifying agents, solution promoters, salts for regulatingthe osmotic pressure and/or buffers. In addition, they may also containother therapeutically valuable substances. The compositions are preparedaccording to conventional mixing, granulating or coating methods,respectively, and contain about 0.1 to 75%, in another embodiment, thecompositions contain about 1 to 50%, of the active ingredient.

For example, injectable solutions are produced using solvents such assesame or peanut oil or aqueous propylene glycol, as well as aqueoussolutions of water-soluble pharmaceutically-acceptable salts of thecompounds. In some embodiments, dispersions are prepared in glycerol,liquid polyethylene glycols and mixtures thereof in oils. Under ordinaryconditions of storage and use, these preparations contain a preservativeto prevent the growth of microorganisms. The terms “parenteraladministration” and “administered parenterally” as used herein meansmodes of administration other than enteral and topical administration,usually by injection, and includes, without limitation, intravenous,intramuscular, intraarterial, intrathecal, intracapsular, intraorbital,intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous,subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal andintrasternal injection and infusion.

For rectal administration, suitable pharmaceutical compositions are, forexample, topical preparations, suppositories or enemas. Suppositoriesare advantageously prepared from fatty emulsions or suspensions. Thecompositions may be sterilized and/or contain adjuvants, such aspreserving, stabilizing, wetting or emulsifying agents, solutionpromoters, salts for regulating the osmotic pressure and/or buffers. Inaddition, they may also contain other therapeutically valuablesubstances. The compositions are prepared according to conventionalmixing, granulating or coating methods, respectively, and contain about0.1 to 75%, in another embodiment, compositions contain about 1 to 50%,of the active ingredient.

In some embodiments, the compounds are formulated to deliver the activeagent by pulmonary administration, e.g., administration of an aerosolformulation containing the active agent from, for example, a manual pumpspray, nebulizer or pressurized metered-dose inhaler. In someembodiments, suitable formulations of this type also include otheragents, such as antistatic agents, to maintain the disclosed compoundsas effective aerosols.

A drug delivery device for delivering aerosols comprises a suitableaerosol canister with a metering valve containing a pharmaceuticalaerosol formulation as described and an actuator housing adapted to holdthe canister and allow for drug delivery. The canister in the drugdelivery device has a headspace representing greater than about 15% ofthe total volume of the canister. Often, the polymer intended forpulmonary administration is dissolved, suspended or emulsified in amixture of a solvent, surfactant and propellant. The mixture ismaintained under pressure in a canister that has been sealed with ametering valve.

For nasal administration, either a solid or a liquid carrier can beused. The solid carrier includes a coarse powder having particle size inthe range of, for example, from about 20 to about 500 microns and suchformulation is administered by rapid inhalation through the nasalpassages. In some embodiments where the liquid carrier is used, theformulation is administered as a nasal spray or drops and includes oilor aqueous solutions of the active ingredients.

The active reagents can be prepared with carriers that will protectagainst rapid elimination from the body. For example, a controlledrelease formulation can be used, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

The compositions and formulations of the instant invention can alsocomprise one or more desiccants. Suitable desiccants that can be used inthe present invention are those that are pharmaceutically safe, andinclude, for example, pharmaceutical grades of silica gel, crystallinesodium, potassium or calcium aluminosilicate, colloidal silica,anhydrous calcium sulphate and the like. The desiccant may be present inan amount from about 1.0% to 20.0%, or from about 2% to 15% w/w (or anyvalue within said range).

Also contemplated are formulations that are rapidly dispersing dosageforms, also known as “flash dose” forms. In particular, some embodimentsof the present invention are formulated as compositions that releasetheir active ingredients within a short period of time, e.g., typicallyless than about five minutes, in another embodiment, less than aboutninety seconds, in another embodiment, less than about thirty secondsand in another embodiment, in less than about ten or fifteen seconds.Such formulations are suitable for administration to a subject via avariety of routes, for example by insertion into a body cavity orapplication to a moist body surface or open wound.

Typically, a “flash dosage” is a solid dosage form that is administeredorally, which rapidly disperses in the mouth, and hence does not requiregreat effort in swallowing and allows the compound to be rapidlyingested or absorbed through the oral mucosal membranes. In someembodiments, suitable rapidly dispersing dosage forms are also used inother applications, including the treatment of wounds and other bodilyinsults and diseased states in which release of the medicament byexternally supplied moisture is not possible.

“Flash dose” forms are known in the art; see for example, effervescentdosage forms and quick release coatings of insoluble microparticles inU.S. Pat. Nos. 5,578,322 and 5,607,697; freeze dried foams and liquidsin U.S. Pat. Nos. 4,642,903 and 5,631,023; melt spinning of dosage formsin U.S. Pat. Nos. 4,855,326, 5,380,473 and 5,518,730; solid, free-formfabrication in U.S. Pat. No. 6,471,992; saccharide-based carrier matrixand a liquid binder in U.S. Pat. Nos. 5,587,172, 5,616,344, 6,277,406,and 5,622,719; and other forms known to the art.

The compounds of the invention are also formulated as “pulsed release”formulations, in which the compound is released from the pharmaceuticalcompositions in a series of releases (i.e., pulses). The compounds arealso formulated as “sustained release” formulations in which thecompound is continuously released from the pharmaceutical compositionover a prolonged period.

Also contemplated are formulations, e.g., liquid formulations, includingcyclic or acyclic encapsulating or solvating agents, e.g.,cyclodextrins, polyethers, or polysaccharides (e.g., methylcellulose),or in another embodiment, polyanionic β-cyclodextrin derivatives with asodium sulfonate salt group separate from the lipophilic cavity by analkyl ether spacer group or polysaccharides. In one embodiment, theagent is methylcellulose. In another embodiment, the agent is apolyanionic β-cyclodextrin derivative with a sodium sulfonate saltseparated from the lipophilic cavity by a butyl ether spacer group,e.g., CAPTISOL® (CyDex, Overland, Kans.). One skilled in the art canevaluate suitable agent/disclosed compound formulation ratios bypreparing a solution of the agent in water, e.g., a 40% by weightsolution; preparing serial dilutions, e.g. to make solutions of 20%, 10,5%, 2.5%, 0% (control), and the like; adding an excess (compared to theamount that can be solubilized by the agent) of the disclosed compound;mixing under appropriate conditions, e.g., heating, agitation,sonication, and the like; centrifuging or filtering the resultingmixtures to obtain clear solutions; and analyzing the solutions forconcentration of the disclosed compound.

All publications and patent documents cited herein are incorporatedherein by reference as if each such publication or document wasspecifically and individually indicated to be incorporated herein byreference. Citation of publications and patent documents is not intendedas an admission that any is pertinent prior art, nor does it constituteany admission as to the contents or date of the same. The inventionhaving now been described by way of written description, those of skillin the art will recognize that the invention can be practiced in avariety of embodiments and that the foregoing description and examplesbelow are for purposes of illustration and not limitation of the claimsthat follow.

EXAMPLES Example 1 Small Scale Synthesis of KX2-391

The preliminary synthesis described below was illustrated inUS20060160800A1. This procedure is useful for small scale reactions, forexample, reactions that produce up to 50 g of product.

For the following synthesis, unless otherwise noted, reagents andsolvents were used as received from commercial suppliers. Proton andcarbon nuclear magnetic resonance spectra were obtained on a Bruker AC300 or a Bruker AV 300 spectrometer at 300 MHz for proton and 75 MHz forcarbon. Spectra are given in ppm (δ) and coupling constants, J, arereported in Hertz. Tetramethylsilane was used as an internal standardfor proton spectra and the solvent peak was used as the reference peakfor carbon spectra. Mass spectra and LC-MS mass data were obtained on aPerkin Elmer Sciex 100 atmospheric pressure ionization (APCI) massspectrometer. LC-MS analyses were obtained using a Luna C8(2) Column(100×4.6 mm, Phenomenex) with UV detection at 254 nm using a standardsolvent gradient program (Method B). Thin-layer chromatography (TLC) wasperformed using Analtech silica gel plates and visualized by ultraviolet(UV) light, iodine, or 20 wt % phosphomolybdic acid in ethanol. HPLCanalyses were obtained using a Prevail C18 column (53×7 mm, Ailtech)with UV detection at 254 nm using a standard solvent gradient program(Method A or B).

Method A: A = Water with 0.1 v/v Trifluoroacetic Acid B = Acetonitrilewith 0.1 v/v Trifluoroacetic Acid Time Flow (min) (mL/min) % A % B 0.03.0 95.0 5.0 10.0 3.0 0.0 100.0 11.0 3.0 0.0 100.0

Method B: A = Water with 0.02 v/v Trifluoroacetic Acid B = Acetonitrilewith 0.02 v/v Trifluoroacetic Acid Time Flow (min) (mL/min) % A % B 0.02.0 95.0 5.0 4.0 2.0 5.0 95.0

Synthesis of N-benzyl-2-(5-bromopyridin-2-yl)acetamide

A flask was charged with5-(5-bromopyridin-2(1H)-ylidene)-2,2-dimethyl-1,3-dioxane-4,6-dione(1.039 g, 3.46 mmol), benzylamine (0.50 mL, 4.58 mmol), and toluene (20mL). The reaction was brought to reflux under nitrogen for 18 hours,then cooled and placed in a freezer until cold. The product wascollected by filtration and washed with hexanes to yield a mass ofbright white crystals (1.018 g, 96%).

Synthesis of4-(2-(4-(4,4,5,5-tetramethyl[1,3,2]dioxaborolan-2-A-phenoxy)ethyl)morpholine

To a stirring solution of4-(4,4,5,5-tetramethyl[1,3,2]dioxaborolan-2-yl)-phenol (2.55 g, 11.58mmol), 2-morpholin-4-ylethanol (1.60 mL, 1.73 g, 13.2 mmol) andtriphenyl phosphine (3.64 g, 13.9 mmol) in methylene chloride (60 mL) at0° C. was added dropwise DIAD (2.82 g, 13.9 mmol). The reaction wasallowed to warm to room temperature and stir overnight. After 18 hours,additional portions of triphenyl phosphine (1.51 g, 5.8 mmol),2-morpholin-4-ylethanol (0.70 mL, 5.8 mmol), and DIAD (1.17 g, 5.8 mmol)were added. After stirring an additional 2 hours at room temperature thereaction was concentrated and the residue purified by flashchromatography (5% to 25% EtOAc in CHCl₃) to provide the product as awhite solid (2.855 g, 74%).

Synthesis of2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamideKX2-391

A 10 mL reaction tube with a septum closure and stir bar was chargedwith N-benzyl-2-(5-bromopyridin-2-yl)acetamide (123 mg, 0.403 mmol),4-(2-(4-(4,4,5,5-tetramethyl[1,3,2]dioxaborolan-2-yl)-phenoxy)ethyl)morpholine(171 mg, 0.513 mmol), and FibreCat 1007¹ (30 mg, 0.015 mmol). Ethanol (3mL) was added, followed by aqueous potassium carbonate solution (0.60mL, 1.0 M, 0.60 mmol). The tube was sealed and heated under microwaveconditions at 150° C. for 10 minutes. The reaction was cooled andconcentrated to remove the majority of the ethanol, and then taken up in10 mL of ethyl acetate and washed successively with water and saturatedsodium chloride solution. The organic layer was dried with MgSO₄,filtered and concentrated to a white solid. This white solid wastriturated with ethyl ether to give KX2-391 as a white solid (137 mg,79%): mp 135-137° C.; ¹H NMR (300 MHz, CDCl₃) δ 8.70 (d, 1H, J=2.0 Hz),7.81 (dd, 1H, J=2.4 Hz, J=8.0 Hz), 7.65 (br s, 1H), 7.49 (d, 2H, J=8.8Hz), 7.37-7.20 (m, 6H), 7.01 (d, 2H, J=8.8 Hz), 4.49 (d, 2H, J=5.8 Hz),4.16 (t, 2H, J=5.7 Hz, 3.82 (s, 2H), 3.78-3.72 (m, 4H), 2.84 (t, 2H,J=5.7 Hz), 2.62-2.58 (m, 4H); HPLC (Method B) 98.0% (AUC), t_(R)=1.834min.; APCI MS m/z 432 [M+H]⁺. ¹ Polymer bounddi(acetato)dicyclohexylphenylphosphinepalladium(II), manufactured byJohnson Matthey, Inc. and available from Aldrich (catalog #590231).

Example 2 Intermediate Scale Synthesis of KX2-391 di-hydrochloride

The synthesis outlined in this example can be used on intermediate-scalereactions. The preparation of batches of at least 50 g of thedihydrochloride salt of KX2-391 is shown in Scheme 1. The linearsynthesis consisted of 6 steps, a seventh step being the preparation ofone of the reagents, 6-fluoropyridin-3-ylboronic acid (which is alsoavailable commercially). The overall yield of the sequence was 35% withan average yield of 83%, with the lowest yielding step being that of68%. Of the seven steps only one required chromatography. The procedurelisted below was performed on a 70 g scale.

The first step is a Williamson ether synthesis between 4-bromophenol(131 g) and N-chloroethylmorpholine (1 as the HCl salt; 141 g) usingK₂CO₃ powder (3 to 3.5 equivalents) as the base and having acetonitrileas the solvent. The ingredients were mixed and stirred at refluxovernight with high conversion (96.3-99.1%). After dilution withdichloromethane and heptane, the reaction mixture was filtered andevaporated to give the desired product 2 in essentially a quantitativeyield (216 g). Note that with similar substrates (e.g.,4-bromo-3-fluorophenol), conversions (even with extensive heating) werenot always so high (e.g., 59.9-98.3%). Both the alkyl chloride and theK₂CO₃ are preferably purchased from Aldrich. If continued heating doesnot drive reaction to completion, unreacted bromophenol can readily beremoved by dissolving the crude reaction mixture in 4 parts toluene andwashing out the phenol with 4 parts 15% aqueous NaOH.

One of the reagents required for the second step (Suzuki coupling) was6-tluoropyridin-3-ylboronic acid (4). Although available commercially,this reagent was readily prepared by lithium-bromide exchange of5-bromo-2-fluoropyridine (3, 102 g) with n-butyllithium (1.2 eq) at lowtemperatures (<−60° C.) in TBME followed by the addition oftriisopropylborate (1.65 eq). Both stages of the reaction are brief,with an overall reaction time (including addition times) of ˜3 h.Quenching is achieved with aqueous 24% NaOH, which also extracts theproduct leaving impurities in the organic layer. Once the aqueous layeris removed, it is then neutralized with HCl and extracted with EtOAc.After drying the organics and diluting with some heptane, concentrationleads to precipitation/crystallization of the product. Filtration gavethe boronic acid 4 in relatively high purity (96.4% AUC) and good yield(69 g, 79-90%; see note on estimation of yield in the experimentalsection), which can be used without further purification.

The second reaction step in the linear sequence (a Suzuki coupling) is asimple reaction to set up; all the reagents [2 (111 g), aqueous Na₂CO₃,DME, and Pd(PPh₃)₄ (0.04 eq)] were charged to the reaction flask and themixture heated at reflux; note that the reaction mixture was degassed toremove oxygen. Once the reaction is complete (within 7 h), the work-upinvolved decanting (or siphoning off) of reaction solution from theorganic salts on the side of the flask (there was no visible aqueouslayer), the flask was rinsed, and dried, and the solvent was removedfrom the combined organics. Crystallization of crude 5 fromisopropanol/heptane provided material of improved purity compared to thecrude, but still required chromatography (ratio of silica gel to crudewas ˜8.5:1) to obtain material of adequate purity (>98%); the yield was68% (79.5 g). Use of clean 5 prevented the need for chromatography inthe next step, acetonitrile displacement of the fluorine atom.

The replacement of fluoride with acetonitrile was also a simplereaction, and a simple room temperature crystallization of the crudeproduct provided clean 6 in high yield and purity. The reaction involvedinitial formation of the “enolate” from acetonitrile (6.5 eq) usingpotassium hexamethyldisilane KHMDS (8 eq)/THF at −10° C. followedimmediately by the addition of fluoride 5 (79 g). The reaction was quickand after one hour quenching was achieved with saturated brine. Afterdrying and evaporation of solvent of the organics, the resulting crudemixture consisted of only two components, the desired product and a muchless polar product from apparent self-condensation of acetonitrile. Thecrude mixture was swirled in isopropanol/heptane and allowed to sitovernight, which resulted in complete crystallization of the product,which was filtered off and washed to provide high purity 6 (99.3% AUC)in good yield (64 g, 76%).

Methanolysis of 6 (64 g) was accomplished by heating in 40% H₂SO₄ (inMeOH) until the reaction was complete (25 h). The reaction was thencooled, stirred with MgSO₄ to convert traces of hydrolyzed product(ArCH₂—CO₂Me) back to product, and then added to cooled, aqueous K₂CO₃,with simultaneous extraction into dichloromethane. Drying andevaporation of most of the DCM followed by addition of 5% EtOAc (inheptane) and further concentration resulted in the crystallization ofthe product. Filtration of the solid and washing gave high purity (98.9%AUC) 7 in good yield (82%), additional high purity product (4 g) beingobtained from the mother liquors for a total yield of 61.7 g (87%).

The amidation step also involved charging of the reaction vessel withthe ingredients (7 (61 g), benzyl amine (3 eq), and high boilinganisole) and then heating at reflux until the reaction was complete.Cooling of the reaction mixture resulted in complete crystallization ofthe target compound with high purity (98.9%) and good yield (81%).

The final step was the formation of the dihydrochloric salt of thetarget compound. In order to ensure complete protonation at both basicsites, the reaction was conducted in absolute ethanol, which freelydissolved the dihydrochloride salt. After evaporation to near dryness,the reaction mixture was “chased” with ethanol twice to remove excesshydrogen chloride. The resulting viscous oil was dissolved in ethanol (2parts) and then added, with rapid stirring, to a large volume (20 parts)EtOAc (ethyl acetate). Filtration, washing with ethyl acetate (noheptane) and vacuum drying provided the dihydrochloride salt of KX2-391as a creamy-white powder. A total of 68 g (yield of 97%) was obtained ofthe final salt in high purity (99.6% AUC), which contained traces ofEtOAc (4.8% w/w), EtOH (0.3% w/w), and heptane (0.6% w/w; from a finalwash with heptane prior to vacuum drying). This salt was alsocrystallized (instead of the precipitation method described above) fromhot EtOH/EtOAc to afford crystalline beads that had much lower entrappedsolvent levels (only 0.26% w/w of EtOAc and 0.45% w/w of EtOH) and wasfree-flowing.

Preparation of 4-(2-(4-bromophenoxy)ethyl)morpholine (2)

A 5 L three-necked round-bottomed flask, equipped with mechanicalstirrer, thermometer with adapter, condenser, and nitrogen inlet (on topof condenser), was charged with 1 (140.7 g, 0.756 mol), 4-bromophenol(130.6 g, 0.755 mol), anhydrous K₂CO₃ powder (367.6 g, 2.66 mol, 3.5eq), and acetonitrile (1.3 L). The mixture was vigorously stirred (bladetouching bottom of flask) at 80° C. (overnight), followed by dilutionwith DCM (500 mL) and heptane (200 mL) and filtration through Celite.Evaporation to dryness (rotovap, then high vac) gave 2 as a light yellowoil (216.00 g, yield of 100%, 96.3% AUC, contains 3.7% unreactedbromophenol). This material was used successfully without furtherpurification.

¹H NMR (CDCl₃) δ 2.57 (t, 4H), 2.79 (t, 2H), 3.73 (t, 4H), 4.08 (t, 2H),6.78 (d, 2 H), 7.37 (d, 2H). MS (from LC/MS): m/z 287.1 [M+1].

That the bromophenol can be readily removed was demonstrated on a 2 gsample by first dissolving the sample in toluene (8 g) and washing with8 g of 15% aqueous NaOH; liquid chromatography showed no trace ofunreacted bromophenol in the recovered product (1.97 g; 98.5% recovery).

Preparation of 6-fluoropyridin-3-ylboronic acid (4)

To stirred and cooled (dry ice-acetone bath) anhydrous [TBME] (620 mL;in a 3 L three-necked round-bottomed flask equipped with mechanicalstirrer, temperature probe with adapter, and nitrogen inlet) was added(via syringe) 2 M BuLi (352 mL, 0.704 mol, 1.2 eq). To this rapidlystirred and cooled (<−75° C.) mixture was added a solution of 3 (102.2g, 0.581 mol) in anhydrous TBME (100 mL) over a period of 13 min duringwhich time the internal temperature rose to −62° C. The reaction wasstirred for another 45 min (the temperature was maintained between −62°C. and −80° C.), followed by the rapid and sequential addition of fourportions of triisopropylborate (total of 180 g, 0.957 mol, 1.65 eq). Atthe end of the addition the internal temperature had risen to −33° C.After stirring an additional 45 min over the cold bath (internaltemperature lowered from −33° C. to −65° C.), the cold bath was removedand the stirred mixture on its own rose to −22° C. over a period of 50min. After warming (via water bath) to 6° C. over a period of 15 min,the stirred reaction mixture was placed in an ice-water bath and thenquenched under nitrogen with a cooled solution of NaOH (160 g) in water(500 mL) Once the addition was complete, the internal temperature was20° C. This mixture was stirred at room temperature for 1.5 h. Theaqueous layer was removed, neutralized to pH 7 with ˜350 mL concentratedHCl, and then extracted with EtOAc (3×1 L). Because the pH was now 8-9,the aqueous layer was adjusted to pH 7 using ˜15 mL concentrated HCl andextracted further (2×1 L) with ethyl acetate. The combined EtOAcextracts were dried (Na₂SO₄), filtered, and concentrated to a volume of˜150 mL. With swirling of the concentrate, heptane was added in portions(total volume of 300 mL) resulting in the precipitation/crystallizationof the product. Filtration, washing of the solid with heptane (100 mL,300 mL, then another 300 mL), and air drying gave the title product asan off-white solid (68.6 g, yield of 79-90%*; LC purity of 96.4%, NMRshowed an estimated 5.5% w/w of heptane), which was used successfullywithout further purification. LC/MS showed it to be a mixture of the twofollowing entities, the intensity of the higher molecular weight entitybeing major (*Note: yield of reaction is 79% if the boronic acid isassumed to be the only constituent and is 90% if it is assumed that thecyclic borate is the only constituent):

hu 1H NMR (CDCl₃) δ 7.14 (dd, 1H), 8.27 (ddd, 1H), 8.39 (br s, 2H, 2OH), 8.54 (fine d, 1H). MS (from LC/MS): m/z 143.0 [M+1; for boronicacid] and 370.0 [M+1; for cyclic borate above].

Preparation of 4-(2-(4-(6-fluoropyridin-3-yl)phenoxy)ethyl)morpholine(5)

A 2 L three-necked round-bottomed flask equipped with mechanicalstirrer, thermometer and adapter, condenser, and nitrogen inlet (at topof condenser) was charged with 2 (110.7 g, 0.387 mol), 4 (71.05 g, 0.477mol, 1.23 eq) and DME (700 mL). The resulting stirred solution wasdegassed by passing a rapid stream of nitrogen through the stirredsolution over a period of 5 min followed by the addition of a degassedsolution of Na₂CO₃ (121.06 g, 1.142 mol, 3 eq) in H₂O (250 mL) and alsosolid Pd(PPh₃)₄ (19.8 g, 0.044 eq). Immediately after the last addition,the head space above the reaction mixture was purged with nitrogen andthe mixture then stirred at 80-85° C. (internal temperature) for 7 h,followed by cooling to room temperature. Because of the lack of anaqueous layer, the supernatant was decanted, leaving behind theinorganic salts (with adsorbed water). The reaction flask with theinorganic salts was washed with 50% dichloromethane/ethyl acetate (2×250mL), the washes being added to the decanted supernatant. These combinedorganics were dried (Na₂SO₄), filtered, and evaporated to dryness to adark brown oil (148 g). To this oil was added 150 g of 50%heptane/isopropyl alcohol (IPA) and after swirling and cooling (via icewater bath), crystallization began. Additional heptane (50 g) was addedand the resulting solid was filtered, washed, and air dried to give 48 gof a light brown solid. After evaporating the filtrate to dryness, theresulting mixture was swirled in 100 mL of 50% heptane/IPA followed bythe addition of more heptane (˜100 mL), stoppering and placing in thefreezer for crystallization. The resulting solid was filtered, washedwith heptane, and air dried to give 61 g of a gummy solid. Evaporationof the resulting filtrate gave an oil (34 g) which contained significantless polar impurities including Ph₃P═O and so it was partitioned between2 N HCl (240 mL) and EtOAc (220 mL). The bottom aqueous layer wasremoved and then stirred with EtOAc while neutralizing with K₂CO₃ to apH of 7-8. The EtOAc layer was dried, filtered, and evaporated todryness (22 g). The 48 g, 61 g, and 22 g portions were chromatographedover silica gel (1.1 Kg) packed in DCM. Elution with DCM (400 mL), 50%DCM/EtOAc (5 L), and then 50% DCM/EtOAc (8 L) containing increasingamounts of MeOH/Et₃N (beginning with 1.5% MeOH/1% Et₃N and ending with5% MeOH/3% Et₃N) gave 77.68 g of a viscous oil (purity 98.0%) whichimmediately crystallized upon swirling in heptane (300 mL). Filtration,washing with heptane and air drying gave 75.55 g (98.7% AUC) of solid 5.Additional pure 5 (total of 3.9 g, 98.6-99.3% AUC) was obtained fromearlier chromatographic fractions containing Ph₃P═O by cleaning them upas done for the above 34 g sample, followed by evaporativecrystallization. The total yield of 5 was 79.5 g (68%).

hu 1H NMR (CDCl₃) δ 2.59 (t, 4H), 2.84 (t, 2H), 3.75 (t, 4H), 4.16 (t,2H), 6.97 (dd, 1H), 7.01 (d, 2H), 7.46 (d, 2H), 7.92 (ddd, 1H), 8.37(fine d, 1H). MS (from LC/MS): m/z 303.2 [M+1].

Preparation of2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)acetonitrile (6)

A 3 L three-necked round-bottomed flask was equipped with mechanicalstirrer, thermometer and adapter, additional funnel, and nitrogen inlet(on top of addition funnel, positive pressure through a bubbler). With arapid stream of nitrogen going through the bubbler, the stopper wasremoved and the flask was charged with KHMDS (415.8 g, 2.08 mol) andthen anhydrous THF (1 L). To the stirred and cooled (ice/methanol bath,internal temperature of solution was −8° C.) KHMDS/THF solution wasadded dropwise a solution of MeCN (70 g) in THF (110 mL) over a periodof 22 min followed immediately by the relatively rapid (4 min) additionof a solution of 5 (79.06 g, 0.262 mol) in THF (400 mL), after whichtime the internal temperature of the reaction mixture had reached 10° C.With continued cooling (1 h) the internal temperature was −6° C. and byTLC the reaction appeared complete. After an additional 30 min (internaltemperature of −3° C.), the reaction mixture was quenched with saturatedbrine (1 L) and diluted with EtOAc (500 mL). After removing the aqueouslayer, the organic solution was dried (Na₂SO₄), filtered, and evaporatedto dryness (to an oil) followed by completely dissolving in IPA (150mL), diluting with heptane (300 mL), adding seed crystals (prepared bydissolving ˜100 mg of crude oil in IPA (˜150 mg) and diluting withheptane (˜2.5 mL)), and allowing to stand overnight. After stirring tobreak up the crystalline solid, the solid was filtered, washed with 250mL 2:1 heptane/IPA and then multiple washes with heptane and air driedto give 64.38 g (yield of 76%) of title product 6 as a crystalline tansolid (LC purity of 99.3%). Another 5.88 g of less pure material wasobtained from the filtrate.

hu 1H NMR (CDCl₃) δ 2.59 (t, 4H), 2.84 (t, 2H), 3.74 (t, 4H), 3.97 (s,2H), 4.17 (t, 2H). 7.02 (d, 2H), 7.46 (d, 1H), 7.51 (d, 2H), 7.87 (dd,1H), 8.77 (fine d, 1H). MS (from LC/MS): m/z 324.4 [M+1].

Preparation of methyl2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)acetate (7)

A 2 L single-necked round-bottomed flask was charged with 6 (64.00 g,0.198 mol) and MeOH (360 g) followed by the slow, careful, and dropwiseaddition of H₂SO₄ (240 g) and the resulting homogeneous solution stirredat reflux (115° C. oil bath) until the reaction was complete (25 h with0.8% unreacted starting material) with 3.5% ArCH₂CO₂H. After briefcooling, MgSO₄ (75 g) was added and the mixture swirled and allowed tostand an additional 45 min (composition now 96.3% product, 0.8%unreacted starting material, and 2.5% ArCH₂CO₂H). The reaction mixturewas then added slowly to a rapidly stirred and cooled (ice-water bath)mixture of DCM (2 L) and a solution of K₂CO₃ (450 g) in H₂O (600 mL).The resulting emulsion was allowed to stand overnight. The clearportions of organic solution were siphoned off and the remainderportions were treated iteratively with water and DCM, the clear organicsbeing combined with the original portion that was siphoned off. Thecombined organics were dried (Na₂SO₄), filtered, and concentrated to avolume of ˜1.2 L followed by the addition of 300 mL of 5% EtOAc (inheptane) and then heptane (300 mL) and the mixture concentrated (rotovapwith heat) again to remove the DCM. At this point 15 mL EtOAc was addedand the hot mixture swirled until crystallization had begun, swirlingcontinued until crystallization was near complete, and then allowed tostand and cool to room temperature for complete crystallization. Thesolid was then filtered, washed with 300 mL 5% EtOAc (in heptane) andheptane (100 mL) and then fully air dried to give 57.74 g (yield of 82%)of 7 as a light yellow solid (98.9% AUC). Another 3.94 g of cleanproduct (97.9% AUC) was obtained from the filtrate (total yield of 87%).

hu 1H NMR (CDCl₃) δ 2.60 (t, 4H), 2.84 (t, 2H), 3.74 (overlapping t ands, 6H), 3.89 (s, 2H), 4.17 (t, 2H), 7.01 (d, 2H), 7.34 (d, 1H), 7.49 (d,2H), 7.80 (dd, 1H), 8.74 (fine d, 1H). MS (from LC/MS): m/z 357.4 [M+1].

Preparation of2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamide(KX2-391 free base)

A 1 L single-necked round-bottomed flask was charged with 7 (61.4 g,0.172 mol), benzyl amine (55.6 g, 0.519 mol, 3 eq), and anhydrousanisole (300 g) and then stirred at reflux until reaction wasessentially complete (23 h, 165° C. oil bath temperature; internaltemperature was 147° C.) and then allowed to cool to near roomtemperature. A portion (1 mL) of the reaction mixture was diluted withtoluene (1 mL) resulting in the complete crystallization of thatportion. This seed was then added to the reaction mixture and allowed tostand until the whole reaction mixture had crystallized to a singleblock. Toluene (150 mL) was added and the mixture swirled to break upthe solid. Heptane/toluene (1:1, 100 mL) was added and the solid mixturebroken up further. Finally, heptane (50 mL, then 25 mL) was added andthe mixture broken up even further, allowing to stand an additional 30min before filtering the solid. Filtration of the solid, washing with2:1 toluene/heptane (300 mL), 1:2 toluene/heptane (300 mL), and thenheptane (2×300 mL), and then drying (air, then high vac) gave 60.16 g(yield of 81%) of title product as a white solid (≧98.9% AUC). Another2.5 g of less pure (97.4%) material was obtained from the motherliquors.

hu 1H NMR (CDCl₃) δ 2.60 (t, 4H), 2.83 (t, 2H), 3.74 (t, 4H), 3.82 (s,2H), 4.18 (t, 2H), 4.49 (d, 2H), 7.01 (d, 2H), 7.2-7.35 (m, 6H), 7.49(d, 2H), 7.64 (br t, 1H), 7.81 (dd, 1H), 8.69 (fine d, 1H). MS (fromLC/MS): m/z 432.5 [M+1].

Preparation of4-(2-(4-(6-(2-(benzylamino)-2-oxoethyl)pyridinium-3-yl)phenoxy)ethyl)-morpholin-4-iumchloride (KX2-391, diHCl salt)

To a stirred suspension of KX2-391 (free base, 60.00 g) in absolute EtOH(600 mL) was added 170 mL of 2.5 M HCl (in ethanol), 25 mL EtOH beingadded to wash down the sides of the flask. The resulting homogeneoussolution was stirred at room temperature (20 min) and then evaporated tonear dryness (to frothing). After chasing with EtOH (2×150 mL), theresidue was taken up again in EtOH (150 mL) and then was followed by theslow addition of heptane until the mixture appeared saturated (33 mLrequired for cloudiness to remain). After sitting overnight, two layershad formed. After adding additional heptane (250 mL) crystallizationstill could not be induced and so the reaction mixture was concentratedto a volume of ˜200 mL at which time the mixture was homogeneous. Thisthick homogeneous solution was added dropwise to very rapidly stirred(mechanical) EtOAc (2 L). After the addition was complete, a 25 mL EtOHrinse of the original flask and addition funnel was added to the rapidlystirred mixture. The rapid stirring was continued for another ˜1 h andthen the mixture was filtered and the solid (partly gummy) was washedwith EtOAc (300 mL) and then heptane. As soon as the heptane wash began,the solid got much gummier. The fitted Buchner funnel and its contentswere covered (paper towel/rubber band) and immediately placed in thevacuum oven. After overnight vacuum at ˜45° C., the vacuum was releasedunder nitrogen, and the Buchner funnel containing the product (foamysolid) was immediately placed in a zip-lock back and then, undernitrogen (glove bag), transferred to a bottle and the foamy solid brokenup (spatula) to a powder. A second night under high vacuum (˜45° C.)resulted in only 1.3 g of additional weight loss. Constant weight wasessentially attained with the third night of high vacuum (˜45° C.) whereonly 0.2 g of weight was lost. The final weight of material was 68.05 g(yield of 97%), containing 0.29 eq (4.8% w/w) of EtOAc, 0.035 eq (0.3%w/w) EtOH, and 0.03 eq (0.6% w/w) heptane. The purity was 99.6%.

¹H NMR (DMSO-d₆) δ 3.1-3.3 (m, 2H), 3.45-3.65 (m, 4H), 3.8-4.0 (m, 4H),4.11 (s, 2H), 4.32 (d, 2H), 4.57 (t, 2H), 7.19 (d, 2H), 7.2-7.4 (m, 5H),7.88 (d, 2H), 7.93 (d, 1H), 8.68 (dd, 1H), 8.99 (br t, 1H), 9.10 (fined, 1H), 11.8 (br s, 1H). MS (from LC/MS): m/z 432.5 [M+1 of free base].

Elemental analysis (for C₂₆H₂₉N₃O₃.2HCl.0.035 EtOH.0.29 EtOAc.0.03heptane.0.8H₂O):

a. Calculated (%): C, 60.03; H, 6.54; N, 7.65; Cl, 12.91

b. Observed (%): C, 59.85/59.97; H, 6.54/6.47; N, 7.67/7.67; Cl,13.10/13.24

Calculated FW: 534.63 (does not take into account the 0.8 H₂O whichprobably arose during handling of this very hygroscopic powder, since ¹HNMR shows no evidence for H₂O).

The ethyl chloride level in this material was measured and found to be98 ppm. The sample was also analyzed and found to contain 5,800 ppm ofheptane.

Analysis of another portion of this sample yielded the followingresults: 99.6% AUC, 1640 ppm ethanol, 41,480 ppm ethyl acetate, 5600 ppmheptane, no anisole detected, and 120 ppm ethyl chloride.

A procedure for recrystallizing the salt was also developed using theabove dried salt. This procedure would work just was well on the highlypure crude salt (containing residual EtOH) obtained from concentratingthe HCl salt-forming reaction mixture:

The salt (575 mg) was dissolved in twice the mass of absolute EtOH(1.157 g) and then heated under nitrogen. To this hot solution (stirred)was added 1.6 g of 25% EtOH (in EtOAc) followed by the addition of EtOAc(0.25 mL) resulting in a cloudiness that remained. The cloudy hotsolution was allowed to cool to room temperature during which timecrystallization occurred. After crystallization was complete (2 h), thecrystalline solid was filtered, washed with anhydrous EtOAc (˜40 mL),and vacuum dried to give 424 mg of the dihydrochloride salt of KX2-391as a free-flowing solid (tiny beads, 99.8% AUC) containing only 0.05 eq(0.45% w/w) of EtOH and 0.015 eq (0.26% w/w) of EtOAc. Slightly betterrecovery (460 mg from 586 mg) was attained using isopropanol/EtOAc butthe level of solvent entrapment was higher [0.085 eq (1.0% w/w) ofisopropanol and 0.023 eq (0.4% w/w) of EtOAc].

Example 3 Large Scale Synthesis of KX2-391 di-HCl

Reagents and solvents were used as received from commercial suppliers.Progress of the reactions was monitored by HPLC, GC/MS, or ¹H NMR.Thin-layer chromatography (TLC) was performed using Analtech silica gelplates and visualized by UV light (254 nm). High pressure liquidchromatography (HPLC) was performed on an Agilent 1100 Seriesinstruments. Proton and carbon nuclear magnetic resonance spectra wereobtained using a Bruker AV 300 at 300 MHz for proton and 75 MHz forcarbon. The solvent peak was used as the reference peak for proton andcarbon spectra.

Preparation of 4-(2-(4-Bromophenoxy)ethyl)morpholine (2)

A 50 L jacketed reactor equipped with a reflux condenser and temperatureprobe was charged with 4-(3-chloropropyl)morpholine (2.44 kg, 0.54 mol),4-bromophenol (2.27 kg, 0.54 mol, 1.0 equiv.), powdered potassiumcarbonate (6.331 kg, 1.88 mol, 3.50 equiv.), and DMF (12.2 L) andstirred. The reaction mixture was then heated to 60-65° C. and stirredovernight. After 17.5 h, the reaction mixture was cooled to 20-25° C.The reaction mixture was charged to a different reactor equipped withbottom valve for the work-up. While maintaining a temperature between20-30° C., DI water (48.7 L) was charged to the reactor. The phases wereseparated. The aqueous layer was extracted with MTBE (3×24.4 L). To thecombined organics, DI water (18.3 L) and then 6M sodium hydroxide (18.2L) were added. The mixture was stirred for 2-5 minutes and the phaseswere separated. The organic phase was washed with water (24.4 L) andbrine (24.4 L), dried over magnesium sulfate, filtered, and concentratedto give 3370 g of a yellow oil (89% crude yield, 99.4% AUC by HPLC).

Preparation of 6-fluoropyridin-3-ylboronic acid (4)

A 72 L reactor equipped with reflux condenser, and temperature probe. Tothe reactor 5-bromo-2-fluoropyridine (1.17 L, 0.568 mol), toluene (18.2L), and triisopropyl borate (3.13 L, 0.68 mol, 1.2 equiv.) were chargedand stirred. Tetrahydrofuran (4.4 L) was added to the reactor and thereaction mixture was cooled to between −35 to −50° C. While maintaininga temperature between −35 to −45° C., n-butyl lithium (2.5 M solution ofhexanes, 5.44 L, 0.68 mol, 1.2 equiv.) was cautiously added to thereactor. After 5 h, the reaction was deemed complete and the reactionmixture was warmed to between −15 to −20° C. To the reaction was added2M HCl (11.80 L) to the reactor while maintaining a temperature between−15° C. and 0° C. The reaction mixture was stirred at 18 to 23° C. for(16 h) and the phases were separated. The organics were then extractedwith 6 M sodium hydroxide (6.0 L). The acidic anbasic aqueous phaseswere mixed in the reactor and 6 M HCl (2.5 L) was added until pH 7.5 wasachieved. Sodium chloride (6.0 kg) was then added to the aqueous phase.The aqueous phase was then extracted with THF (3×20 L). The combinedorganics were dried with magnesium sulfate and concentrated to give 1300g of a tan solid (81% crude yield).

Preparation of 4-(2-(4-(6-fluoropyridin-3-yl)phenoxy)ethyl)morpholine(5)

A 72 L reactor equipped with reflux condenser, sparging tube, bubbler,and temperature probe was charged with 6-fluoropyridin-3-ylboric acid(2.84 kg, 1.24 equiv.), 4-(2-(4-bromophenoxy)ethyl)morpholine (4.27 kg,1.0 equiv.), and DME (27 L). Agitation was started and sodium carbonate(4.74 kg, 3.0 equiv.) as a solution in DI water (17.1 L) was thencharged to the reaction mixture. Argon was bubbled through the reactionmixture for 50 minutes. Under an argon atmosphere,tetrakis(triphenylphosphine)palladium (750 g, 0.04 equiv.) was added tothe reaction mixture as a slurry in DME (1.0 L). The reaction mixturewas heated to 75-85° C. and stirred overnight (17 h). The reactionmixture was cooled to between 18-22° C. DI water (26.681 kg) and MTBE(26.681 L) were charged to the reactor and stirred for 5 minutes. Thephases were separated and the aqueous phase was extracted with MTBE(2×26.7 L). The combined organics were extracted with 2M HCl (1×15.0 L,3×21.8 L). The aqueous phase was then charged back to the reactor andethyl acetate was added (26.7 L). The pH was adjusted to 6.2 using 6 Msodium hydroxide (26.7 L) while maintaining a temperature between 15-25°C. The phases were separated and the aqueous phase was extracted withethyl acetate (2×26.7 L). The combined organics were dried withmagnesium sulfate and concentrated to give 4555 g of a residue (101%crude yield, 67.1% AUC by HPLC).

Purification of 4-(2-(4-(6-fluoropyridin-3-yl)phenoxy)ethyl)morpholine(5)

The crude product (575 g) was purified by silica gel chromatography byeluting with methanol/ethyl acetate/heptane (30% ethyl acetate/heptane,50% ethyl acetate/heptane, 75% ethyl acetate/heptane, 100% ethylacetate, and 5% methanol/ethyl acetate). Concentration of the purefractions by TLC (10% methanol/dichloromethane, R_(f)=0.3) provided 420g of a light brown solid (73% recovery, >99.9% AUC by HPLC).

Preparation of2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)acetonitrile (6)

A 1 M solution of NaHMDS (2.0 L, 5.0 equiv.) in THF was charged to a 5-Lflask and cooled to −20 to −15° C. While maintaining a temperature below−10° C., fluoride (119.7 g, 1.0 equiv.) in THF (500 mL) was charged tothe flask over 20 minutes. Acetonitrile (82.5 mL, 4.0 equiv.) in THF(170 mL) was added to the flask over 20 minutes, while maintaining atemperature below −10° C. The reaction mixture was then stirred for 1 h.To the reaction was added brine (1.5 L, 12.6 vol.) at a rate as tomaintain a temperature below 10° C. The solution was then warmed to roomtemperature and the layers were allowed to separate. The mixture wasfiltered over Celite and washed with THF (1×200 mL, 1×100 mL). Theaqueous phase was extracted with toluene (750 mL). The combined organicswere dried with magnesium sulfate, filtered, washed with toluene (2×250mL), and concentrated to dryness. Toluene (1 L) was added and thesolution was concentrated to dryness again to give 169.8 g of an oil.MTBE (1190 mL, 7 vol.) was added to the oil at 50° C. and stirred for 15minutes. Heptane (850 mL, 5vol.) was added over ten minutes at 50° C.The mixture was then cooled to room temperature over 1.5 h and stirredfor 2 h. The slurry was filtered, washed with 1:4 MBTE/heptane (2×100mL), and dried in an oven overnight at 45° C. to give 102.3 g of anoff-white solid (80% yield, 98.8% AUC by HPLC).

Preparation of methyl2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)acetate (7)

Nitrile 6 (101 g) and methanol (1.01 L, 10 vol.) were charged to a 3-Lflask equipped with stir bar and thermocouple. Concentrated H₂SO₄ (175mL, 10.0 equiv.) was added drop wise to the solution over 15 minuteswhile maintaining a temperature below 60° C. Followed by 30% fumingsulfuric acid (124 mL) was added drop wise to the solution whilemaintaining a temperature below 60° C. The solution was then heated toreflux with a heating mantle and stirred overnight. When the reactionwas deemed complete, it was cooled to 20° C. In a second flask (22 L),saturated sodium bicarbonate (10.7 L) and dichloromethane (1.1 L) werecharged and cooled to 15° C. While maintaining a temperature below 20°C., the reaction mixture was added to the sodiumbicarbonate/dichloromethane mixture. The quench was stirred for 15minutes and the phases were separated. The aqueous phase was extractedwith dichloromethane (1×550 mL, 1×300 mL). The combined organics weredried with magnesium sulfate and concentrated to dryness to give 105 gof an orange solid (94% crude yield, 97.7% AUC by HPLC).

Preparation of2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamide(KX2-391)

Ester 7 (103 g), anisole (513 mL, 5 vol.), and benzylamine (94 mL, 3.0equiv.) were charged to a 3 L flask equipped with thermocouple andoverhead stirrer. The reaction mixture was then heated to 142° C. andstirred for two days. The reaction mixture was cooled to 45-50° C. andstirred for 2 hours. To the mixture was added n-heptane (1.5 L) dropwiseover an hour. The solution was cooled to room temperature over threehours and then stirred overnight. The resulting slurry was filtered,washed with 4:1 Anisole/n-heptane (200 mL) and n-heptane (3×100 mL).Drying in the oven overnight, the resulting product was 112.1 g of a tansolid (90% yield, 99.6% AUC by HPLC). The use of a single isomer ofheptane was essential to adequately quantitate the residual solvent. SeeFIG. 5 for ¹H NMR of KX2-391.

Preparation of2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamidedihydrochloride salt (KX2-391.2HCl)

EtOH (1.0 L) was charged to a 2-L flask and acetyl chloride (62.5 mL,3.0 equiv.) was added slowly to the flask and stirred for 40 minutes.The resulting solution was added to KX2-391 (100 g) over 30 minuteswhile maintaining a temperature of 30° C. The solution was concentratedto a mass of 270 g. The concentrated solution was added to ethyl acetate(2 L) over 20 minutes with rapid stirring. The mixture was stirredovernight and then filtered under nitrogen to give two distinct solidproducts, tan solids (73.5 g) and darker solids (42.2 g). The solidswere dry blended to give a combined yield of 99%. The HPLC analysisindicated 99.0% purity (AUC). Analysis indicated that ethanol waspresent at 2530 ppm, ethyl acetate at 48,110 ppm, ethyl chloride at 170ppm, and no heptane and anisole were detected. Palladium content wasassayed three times and measured to be 29 ppm, 2 ppm, and less than 1ppm.

Crystallization Study of KX2-391.2HCl

The experiments shown in Table 1 were conducted to explore differentcrystallization and precipitation conditions of KX2-391.2HCl.

TABLE 1 Crystallization Study of KX2-391 2HCl Crystallization ConditionsSalt Formation Conditions Nice Amide Solvent EtOAc Temp Solids Expt (g)Lot Solvent Acid (vol) Lot (vol) (C.) (y/n) Comments 02BP097A 0.102BP090D IPA IPA- IPA — 10 60 N Gummy (off- HCl (10)   solids/ white)(5M) slurry formed as EtOAc added 02BP097B 0.1 02BP091E IPA IPA- IPA — —60 N Gummed (white) HCl (10)   out w/ (5M) cooling 02BP097C 0.1 02BP091EIPA IPA- IPA — 6 65 N Dried w/ (white) HCl (15)   EtOAc (5M) first;product oiled out w/ cooling 02BP097D 0.1 02BP091E — IPA- EtOAc/ — — 60N IPA-HCl (white) HCl IPA added to (5M) amide solution; gummed outduring addition (2 drops) 02BP097E 0.3 02BP090D EtOH IPA- EtOH Acros 6.330-60 Y Solids (off- HCl (3.3) observed at white) (5M) 30° C. afterEtOAc added; slow filtering 02BP097F 0.3 02BP093G EtOH IPA- EtOH Acros6.6 60 Y Solids (tan HCl (3.3) observed solid) (5M) during cooling afterEtOAc added; slow filtering 02BP097G 0.3 02BP093G PrOH IPA- PrOH — 1.760 Y Solids (tan HCl (3.3) observed solid) (5M) during cooling afterEtOAc added; slow filtering 02BP097H 0.3 02BP093G BuOH IPA- BuOH — 1.260 Y Solids (tan HCl (5)   observed solid) (5M) during cooling afterEtOAc added; very slow filtering 02BP098A, 1.0 02BP093G EtOH IPA- EtOHAld 4-6 60 N Cloudiness B, C (tan HCl (3.3) observed solid) (5M) earlierthan expected; oiled out 02BP098D 1.0 02BP093G EtOH EtOH—HCl EtOH Ald4.6 60 N Oiled out (tan (2.5M) (3.3) upon solid) cooling 02BP098E 0.302BP090D EtOH EtOH—HCl EtOH Ald 5.3 60 N Oiled out (off- (2.5M) (3.3)from white) EtOAc addition 02BP098F 0.3 02BP091E EtOH IPA- EtOH Acros 660 N Oiled out (white) HCl (3.3) upon (5M) addition of EtOAc 02BP098G0.3 02BP091E PrOH IPA- PrOH — 4 60 N Oiled out (white) HCl (3.3) w/cooling (5M)

Precipitation was achieved by an inverse addition of KX2-391.2HCl in aconcentrated solution of ethanol to a large volume of rapidly stirringethyl acetate. This precipitation procedure was implemented for thedemonstration batch resulting in the formation of two distinct solidtypes. The two distinct solid types were physically separated andfiltered separately. A less dense tan solid (lot 02BP111E, 74 g, 99.1%AUC by HPLC) was filtered first followed by a denser darker solid (lot02BP111F, 43 g, 99.1% AUC by HPLC). After drying in a vacuum oven andbefore blending the two solids a sample of each was retained foranalysis. The data of interest is the Differential Scanning calorimetry(DSC, FIGS. 1 and 2) and X-ray Powder Diffraction (XRPD, FIGS. 3 and 4).The HPLC data for the two samples were comparable while the DSC and XRPDwere different.

Both of the HPLC preparations were greater than 99.0% pure (by area %),the lot 02BP111E sample showed a single endothermic event atapproximately 198° C. while the lot 02BP111F sample showed twoendothermic events at 117° C. and 189° C. The XRPD data for the twosamples were also different the lot 02BP111E sample seemed crystallinewhile the lot 02BP111F sample appeared to be amorphous. The HPLC data,the XRPD data and the DSC data support that the two samples aredifferent forms of the same material.

The two lots of KX2-391.2HCl (lot 02BP111E and 02BP111F) were dryblended resulting in a new lot of KX2-3912HCl (lot 02BP111G).KX2-391.2HCl (lot 02BP111G) contained 170 ppm of ethyl chloride.

Example 4 Preparation of2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamidemesylate (KX2-391.MSA) Preparation of2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)acetonitrile (6)

To round bottom reactor 1 was charged sodium bis(trimethyldisilyl)amide(1.0 M in THF, 23.2 L) and the solution cooled to ≦−10° C. over 52minutes. To a glass carboy, under nitrogen, was charged compound 5 (1400g, 1 wt) and THF (7.0 L, anhydrous, 5 vol)). The batch was stirred withan air powered stirrer under nitrogen. The batch was not completelysoluble and was a hazy solution. The solution of compound 5 was added toreactor 1 over 41 minutes via a 5-L addition funnel. A solution ofacetonitrile (965 mL, anhydrous, 0.69 vol) in THF (2.0 L, anhydrous,1.43 vol) was prepared and added to reactor 1 over 48 minutes at ≦−10°C. via the same addition funnel (a minor amount of a yellow solid waspresent on the reactor wall). After aging for 45 minutes at ≦−10° C. thebatch was sampled for analysis and compound 5 was 0.03% by conversion(specification ≦1.5% by conversion). One hour 24 minutes after sampling,brine (17.6 L, 12.6 vol) was added to reactor 1 over 52 minutes and gavea poorly stirring batch (resembled an emulsion). A pad of diatomaceousearth was prepared on a 24-inch polypropylene funnel (1026 g Celite 545slurried in 3.3 L water with the filtrate discarded). The batch wasfiltered under suction via the pad and the reactor rinsed with THF (1.75L, 1.25 vol) and the rinse transferred to the cake. The cake was rinsedwith a second portion of THF (1.75 L, 1.25 vol) and the total filtrationtime was 1 hour 17 minutes. The filtrate was transferred to reactor 2and the phases separated and held overnight (the batch was held in thereactor under nitrogen). The organic phase (approximately 34.5 L) wasdrained and the aqueous phase extracted with toluene (8.1 L, 5.8 vol),stirring for 16 minutes and settling over 12 minutes. It is possible toomit the toluene extraction and simply add toluene directly to theorganic phase after separation. The aqueous phase (approximately 19 L)was removed and the organic phases combined and dried in reactor 2 withmagnesium sulfate (1400 g, 1 wt, anhydrous) over 55 minutes. The batchwas filtered via a 24-inch polypropylene funnel equipped with an inlinefilter into a glass carboy. The batch was blanketed with argon andstored in the cold room (2-8° C.) pending concentration. The followingday, the batch was concentrated to a residue and rinsed with toluene(11.8 L, 8.4 vol), which in turn was concentrated (water bath 50±5° C.).At the point of the toluene addition, the batch was an orange slurry andremained so after concentration. The total concentration time was 5hours 3 minutes.

To reactor 3 was charged MTBE (13.9 L, 9.9 vol, ACS) which was thenheated to 45±5° C. The MTBE was drained and approximately 2 L of MTBEwas used to slurry the batch from the bulb into reactor 3. The remainingMTBE was added to reactor 3 maintaining the batch at 45±5° C. and thebatch then aged for 33 minutes in this temperature range. n-Heptane (10L, 7.1 vol, 99%) was then added to reactor 3 over 39 minutes maintainingthe batch at 45±5° C. The heat source was disconnected the batch wascooled to 25±5° C. over 4 hours 5 minutes and aged at that temperaturerange for 27 hours 4 minutes. The batch was then filtered under suctionvia a 24-inch polypropylene funnel (PTFE cloth), covered and sucked dryunder nitrogen. The total filtration time was 20 minutes. The orangebatch (net wet weight 1322 g) was dried to constant weight over 48 hours3 minutes in a vacuum oven set at 45±5° C. The batch was transferred totwo 80 oz amber glass jars (Teflon lined closure) and blanketed withargon (1217 g of 6, 81% of theory).

Preparation of methyl2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)acetate (7)

To a 22-L reactor was charged compound 6 (900 g, 2.78 mol) and methanol(9.0 L, 10 vol, anhydrous). Sulfuric acid (1115 mL, fuming) was added tothe suspension over 2 hours 11 minutes to give a dark solution. Themaximum temperature was 65.5° C. (target <65° C.). Sulfuric acid (1565mL, 1.74 vol, concentrated) was added to the batch over 1 hour 49minutes and the batch then heated to visible reflux (74° C.) over 18minutes. The batch was maintained at that temperature for 16 hours 57minutes. The visible gentle reflux was noted to be absent, so the batchwas then heated again to reflux at 79-80° C. over 2 hours 15 minutes.The batch was maintained at that temperature (80±5° C.) for 10 hours 57minutes and the heat source then disconnected; an additional charge ofmethanol (0.75 L, 0.8 vol, anhydrous) was performed after 26 hours 4minutes to replenish the lost solvent volume. It was estimated that2.5-3.3 L of solvent was lost by evaporation. HPLC analysis after 42hours 31 minutes from reflux indicated that the level of compound 6 was0.6% by conversion (specification <1.0%). To each of reactor 1 and 2 wascharged methylene chloride (4.8 L, 5.3 vol) and sodium hydrogencarbonate solution (48 L, 53.3 vol, saturated). The sodium hydrogencarbonate solutions were stored overnight at 2-8° C. and removed thenext morning. Half the batch from the 22-L reactor was added in portionsto each reactor over 47 and 44 minutes respectively (batch temperaturewas 12-13 and 14-15° C., respectively). The quench was accompanied byevolution of carbon dioxide (vigorous at the vortex). The batches fromeach reactor were then transferred to a 200-L reactor and the batchstirred for 16 minutes, then settled over 25 minutes and the organicphase separated. The aqueous phase was extracted successively with twoportions of methylene chloride (5 L, 5.6 vol and 2.7 L, 3 vol); eachextraction took place over 15 minutes stirring with settling over 6 and9 minutes respectively. The combined organic phase was transferred toreactor 3 and dried with magnesium sulfate (900 g, 1 wt, anhydrous) over35 minutes. The batch was then filtered under suction via a 24-inchpolypropylene funnel fitted with Sharkskin cloth and equipped with aninline filter (10 micron, Pall P/N 12077). The filtrate was concentratedon a rotary evaporator over a total of 2 hours 18 minutes at 40±5° C.(water bath temperature). After 54 minutes the batch solidified andformed balls. These were broken up and concentration continued. Thebatch (a mixture of fine solids and brittle chunks) was then furtherground and returned to the bulb and concentration continued. The batchwas transferred to an 80-oz amber jar with a Teflon lined lid andblanketed with argon to give compound 7 (871 g, 88% of theory).

Preparation of2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamide(KX2-391)

To a 22-L reactor was charged compound 7 (650 g, 1.82 mol), anisole(3.25 L, 5 vol, anhydrous) and benzylamine (600 mL, 0.92 vol, 3 equiv).The batch (approximately 18° C.) was heated to 142±5° C. over 1 hour 44minutes, with dissolution occurring at 30° C. The batch was maintainedat 142±5° C. for 69 hours 30 minutes at which point HPLC analysisindicated that compound 7 was 0.9% by conversion (specification ≦1.7% byconversion). The batch was cooled to 45-50° C. over 5 hours 12 minutes(to aid cooling the nitrogen flow was increased once the batch wasapproximately 72° C.). At that temperature range, the batch was poorlystirring and on mixing, the batch temperature increased to 52° C. Itwas >50° C. for ≦15 minutes. The batch was aged for 2 hours 2 minutesonce initially <50° C., then n-heptane (9.75 L, 15 vol, 99%) was addedto the batch over 1 hour 56 minutes, maintaining the batch temperatureat 45-50° C. The heating was then discontinued and the batch cooled to25° C. over 10 hours 32 minutes and then to approximately 20° C. over 20minutes. The total time the batch was maintained ≦25° C. was 4 hours 50minutes (2 hours 47 minutes at approximately 20° C.). The batch wasfiltered under suction via a 24-inch polypropylene filter funnel (fittedwith a PTFE cloth) and the reactor rinsed with anisole/n-heptane (1.3 L,4:1) and the rinse transferred to the cake. The cake was then washedsuccessively with two portions of n-heptane (1.3 L, 0.65 L). The totalfiltration time was 39 minutes. The batch (net wet weight 1004 g ofKX2.391) was transferred to three glass trays and placed into a vacuumoven set at 50° C. and dried to constant weight over 96 hours 26minutes.

Preparation of2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)-N-benzylacetamidemesylate (KX2-391.MSA)

KX2-391 (520 g, 1.21 mol) was transferred to reactor 1 using acetone(41.6 vol, 80 vol, ACS) to facilitate the transfer. The Batch was Heatedto 50±5° C. Over 33 Minutes with dissolution occurring at 30° C. Thebatch was clarified into a second reactor via a transfer pump fittedwith an inline filter (Pall P/N 12077, 10 micron) and reheated from 46°C. to 50±5° C. Methanesulfonic acid (121.4 g, 1.05 equiv, 99% extrapure) was added to the pale yellow batch over 12 minutes and the heatingthen discontinued. After fourteen minutes, white solids were observed,which increased in number to give after 59 minutes a white suspension.The batch was in the range of 25±5° C. after 7 hours 51 minutes and agedfor a further 19 hours 21 minutes (10 hours 30 minutes at ≦27° C.). Thebatch was filtered under suction via a 24-inch polypropylene filter(PTFE cloth) and the reactor rinsed with acetone (2.0 L, clarified, ACS)and the rinse transferred to the cake. The cake was covered with astainless steel cover and sucked dry under a flow of nitrogen. The totalfiltration time was 21 minutes. The batch (net wet weight 764 g) wastransferred to three glass drying trays and dried in a vacuum oven toconstant weight at 25±5° C. over 21 hours 54 minutes (565 g, 89% oftheory). A sample was removed for analysis and the batch maintained invacuo at 25±5° C. The batch was then transferred to two 80-oz amberglass bottles (Teflon lined polypropylene closure), blanketed with argonand stored at −10 to −20° C.

Other Embodiments

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims. It will be understood by thoseskilled in the art that various changes in form and details may be madetherein without departing from the scope of the invention encompassed bythe appended claims.

1. A composition comprising KX2-391mesylate salt.
 2. The composition ofclaim 1, wherein the KX2-391mesylate salt has a purity greater than98.0% as determined by HPLC.
 3. The composition of claim 1, wherein theKX2-391mesylate salt has a purity of 99.0%.
 4. The composition of claim1, wherein the KX2-391mesylate salt has a purity of 99.5%.
 5. Thecomposition of claim 1, wherein the KX2-391mesylate salt has a purity of99.6%.
 6. The composition of claim 1, wherein the KX2-391mesylate salthas a purity of 99.7%.
 7. The composition of claim 1, wherein thecomposition contains less than 2% of an impurity selected from ethylchloride, ethanol, ethyl acetate, heptane, anisole, palladium, andcombinations thereof.
 8. The composition of claim 1 further comprising apharmaceutically acceptable carrier or excipient.
 9. A method oftreating or preventing a disease or disorder in a subject in needthereof comprising administering a composition of claim 1 to saidsubject, wherein the disease or disorder is selected from proliferativedisorders, osteoporosis, cardiovascular disorders, immune systemdysfunction, type II diabetes, obesity, hearing loss, stroke, ophthalmicdisease, atherosclerosis, neuropathic pain, hepatitis B, autoimmunedisease, and transplant rejection.
 10. The method of claim 9, whereinthe disorder is proliferative disorder.
 11. The method of claim 10,wherein the proliferation disorder is pre-cancer or cancer.
 12. Themethod of claim 11, wherein the cancer is colon cancer, lung cancer, orprostate cancer.
 13. The method of claim 9, wherein the disease isophthalmic disease.
 14. The method of claim 13, wherein the ophthalmicdisease is macular degeneration, retinopathy, or macular edema.
 15. Themethod of claim 9, wherein the disease is autoimmune disease.
 16. Themethod of claim 15, wherein the autoimmune disease is rheumatoidarthritis, multiple sclerosis, or lupus.
 17. The method of claim 9,wherein the composition is administered orally.
 18. The method of claim9, wherein the composition is administered topically.